Methods for treatment of diseases

ABSTRACT

We describe peptides and their uses for the treatment of , e.g. acute myocardial infarction and other cytokine storm-associated conditions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/755,693, filed Feb. 27, 2018, which is a 35 USC § 371 national phaseof International Application Number PCT/US2016/048999, filed Aug. 26,2016, which claims benefit of priority under 35 U.S.C. § 119(e) to U.S.Provisional Patent Application Ser. No. 62/211,296 filed Aug. 28, 2015,the contents of which are incorporated herein by reference in theirentirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing, which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 29, 2021, isnamed Sequence Listing.txt and is 18 kilobytes in size.

FIELD OF THE INVENTION

The present disclosure presents novel methods for treatment of diseases,including acute myocardial infarction (AMI); gout; ischemia; stroke;traumatic brain injury; toxic shock, and heart surgery complicationsusing isolated and/or synthesized peptides having unexpectedcytoprotective properties.

BACKGROUND

Acute myocardial infarction (AMI) remains a major cause of morbidity andmortality in the US and worldwide. Despite current strategies for earlyreperfusion, many patients die early during the course, and those whosurvive are at risk for dying later from adverse cardiac remodeling,heart failure, and sudden death.

Numerous publications emphasize the correlation between the size of theinfarct in AMI and the probability to progress to heart failure in thenext 2-5 years post AMI.

Patients presenting with ST-segment elevation (STEMI) are atparticularly high risk for adverse cardiac remodeling, heart failure,and in-hospital and long-term mortality. Although there have beenconsiderable improvements in the treatment of STEMI, the reduction inearly mortality has been associated with an increasing incidence ofheart failure after STEMI. This likely reflects more high risk patientssurviving the index event as well as the aging of the population and theepidemics of hypertension and diabetes. Within 30 days of STEMI, morethan 20% of survivors are diagnosed with heart failure, a diseaseassociated with high morbidity, disability, and mortality.

Heart failure is indeed a major public health problem affectingapproximately 5 million Americans with 500,000 new cases per year. Incontrast to other cardiovascular disease, the incidence and prevalenceof heart failure continue to increase and heart failure is now theleading cause of hospitalization for people aged 65 years, a segment ofthe population that is also rapidly growing. Although survival after theonset of heart failure is also improved, current therapies may slow butnot halt the progression of the disease. With the limitations tofunctional capacity, the progressive symptoms of dyspnea and fatigue,the frequent hospital admissions and the economic consequences of lostproductivity and increasing costs of medical care, heart failure imposesa significant burden on healthcare.

There is an urgent need to develop additional treatments to minimize theinfarct size and prevent heart failure after AMI. The current treatmentin STEMI includes prompt reperfusion of the ischemic myocardium byrestoration of the coronary artery patency (i.e. angioplasty orfibrinolysis), prevention of re-occlusion (i.e. antiplatelet andanticoagulants), and neuro-hormonal blockade (i.e.renin-angiotensin-aldosterone and adrenergic blockers). While each ofthese interventions provide incremental benefit and significantly reducemorbidity and mortality, the incidence of heart failure after STEMI hascontinued to rise, implying that the current treatment paradigm stillmisses one or more key pathophysiologic mechanisms

Determining the mechanisms by which unfavorable cardiac remodeling andheart failure progress despite optimal treatment is thus a critical stepin the search for novel interventions, with the ultimate goal ofreducing the incidence, burden, and mortality of heart failure afterSTEMI.

SUMMARY

We have unexpectedly found that C-terminal peptides from the Serpinmolecule (e.g., short peptide, SP16 (SEQ ID NO: 1)) and variants andderivatives thereof function as potent cytoprotective agents in thetreatment of conditions associated with cytokine storms and/orinfarctions, such as AMI. We have previously shown that these peptideshave anti-inflammatory properties (see, e.g., U.S. Pat. No. 8,975,224),which makes their efficacy in treating conditions associated cell deathdue to cytokine storms and/or infarctions surprising. In the relevantconditions, while uncontrolled inflammation can contribute to thedisease process, inflammation is none the less a critical process whichis required for recovery. This is demonstrated by the fact that manyattempts to modulate the inflammatory response as a treatment for, e.g.,acute myocardial infarction (AMI) have failed.

In contrast, and surprisingly, the peptides described herein 1) permittherapeutic modulation of the inflammatory response, 2) exert acytoprotective effect, and 3) reduce infarct size. This combination ofactivities provides a surprising, and unexpected efficacy in treatingconditions associated with cytokine storms and/or infarctions. Asdescribed in the examples herein, this surprising efficacy is contrastedwith the merely anti-inflammatory activity of, e.g., an IL-1 antagonist.

Accordingly, we provide peptide compositions, pharmaceuticalcompositions comprising the C-terminal Serpin peptides and methods ofusing the peptides to treat conditions including cytokine-stormassociated conditions and/or to reduce infarctions occurring in a numberof conditions, where cytoprotection is desired, such as in conditionsassociated with a risk for ischemia reperfusion injury and resultingpathologies. The unexpected cytoprotective properties of these peptidesallow use of the compositions comprising such peptides to newindications, and allow preventive intervention in conditions associatedwith, e.g., ischemia reperfusion injury.

Specifically, we have shown the novel cytoprotective function of theSP16 peptide consisting of an amino acid sequence VKFNKPFVFLMIEQNTK (SEQID NO: 1) or SP163M VKFNKPFVFLNleIEQNTK in well-established animalmodels for acute myocardial infarction (AMI).

Accordingly, we provide novel uses for a composition comprising anisolated peptide comprising, consisting essentially of, or consisting ofthe amino acid sequence X1-Z1-F-N-K-P-F-X2-Z2-X3-Z3-Q (SEQ ID NO: 2),wherein

X1 is V or L;

X2 is V, L or M or Nle;

X3 is M, Nle, I or V;

Z1 is any amino acid;

Z2 is a sequence of any two amino acids; and

Z3 is a sequence any five amino acids, and wherein the isolated peptideconsists of 37 or fewer amino acids.

The peptide can be modified to extend the shelf life and/orbioavailability using one or more non-natural peptide bonds or aminoacids or by attaching to the peptide functional groups such as, e.g.,polyethylene glycol (PEG).

The composition may further comprise a carrier, such as apharmaceutically acceptable carrier.

In one aspect of any of the embodiments described herein is a methodof 1) treating a disease associated with a cytokine storm, 2) reducingan infarct size, and/or 3) treating acute myocardial infarction (AMI),the method, comprising administering to a human subject affected withthe disease a pharmaceutical composition comprising a peptide selectedfrom the group consisting of:

-   -   (a) a peptide comprising the amino acid sequence

(SEQ ID NO: 2) X1-Z1-F-N-K-P-F-X2-Z2-X3-Z3-Q,wherein

-   -   X1 is V or L;    -   X2 is V, L, Nle, or M;    -   X3 is M, Nle, I or V;    -   Z1 is any amino acid;    -   Z2 is a sequence of any two amino acids; and    -   Z3 is a sequence any five amino acids, and wherein the peptide        comprises 37 or fewer amino acids;    -   (b) a peptide comprising the amino acid sequence

(SEQ ID NO: 1) VKFNKPFVFLMIEQNTK;

-   -   (c) a peptide consisting essentially of the amino acid sequence

(SEQ ID NO: 3) X1-Z1-F-N-X2-P-F-X3-Z2-X4-Z3-X5,wherein

-   -   X1 is V or L;    -   X2 is K or R;    -   X3 is V, L, Nle, or M;    -   X4 is M, Nle, I or V;    -   X5 is K or Q;    -   Z1 is any amino acid;    -   Z2 is a sequence of any two amino acids; and    -   Z3 is a sequence any five amino acids;    -   (d) a peptide consisting essentially of the amino acid sequence

(SEQ ID NO: 4) RFNRPFLR.

-   -   (e) a peptide consisting essentially of the amino acid sequence        of

(SEQ ID NO: 8) RRRFNRPFLRRR.

-   -   (f) a peptide consisting essentially of the amino acid sequence        of

(SEQ ID NO: 1) VKFNKPFVFLMIEQNTK;

-   -   (g) a peptide consisting essentially of the amino acid sequence        of

(SEQ ID NO: 10) FNRPFL;and

-   -   (h) a peptide comprising SEQ ID NO: 57.

In some embodiments of any of the aspects described herein, the diseaseassociated with a cytokine storm is selected from the group consistingof acute myocardial infarction (AMI); gout; stroke; heart surgerycomplications; traumatic brain injury; acute respiratory distresssyndrome (ARDS), sepsis, Ebola, avian influenza, smallpox, and systemicinflammatory response syndrome (SIRS). In some embodiments of any of theaspects described herein, the human subject in need of a reduction ininfarct size is a subject in need of treatment for a disease selectedfrom the group consisting of: acute myocardial infarction (AMI);ischemia; stroke; traumatic brain injury; and toxic shock.

In some embodiments of any of the aspects described herein, thepharmaceutical composition is an oral formulation.

In some embodiments of any of the aspects described herein, the peptidefurther comprises at least one second peptide or protein. In someembodiments of any of the aspects described herein, the at least onesecond protein or peptide is attached to the peptide as a fusionpeptide. In some embodiments of any of the aspects described herein, theat least one second peptide or protein is an epitope tag or a half-lifeextender or both. In some embodiments of any of the aspects describedherein, the peptide comprises one or more D-amino acids.

In some embodiments of any of the aspects described herein, the peptidecauses a 75% decrease in serum TNF-a levels when administered in aneffective amount to a human subject.

In some embodiments of any of the aspects described herein, the peptideconsists of 35 amino acid residues or fewer. In some embodiments of anyof the aspects described herein, the peptide consists of 22 amino acidresidues or fewer. In some embodiments of any of the aspects describedherein, the peptide consists of 21 amino acid residues or fewer.

In some embodiments of any of the aspects described herein, thecomposition further comprises a pharmaceutically acceptable carrier.

In some embodiments of any of the aspects described herein, the subjectdoes not have, does not have symptoms of, or is not diagnosed as havinga condition selected from the group consisting of: type II diabetes,lupus, graft versus host disease, uveitis, eczema, psoriasis, cysticfibrosis, rheumatoid arthritis, acute radiation syndrome, burn patients,inflammatory bowel disease, type I diabetes, and hyperglycemia.

The peptides of the invention can be used to reduce the serum TNF-αlevels in human individuals who have pathologically increased TNF-αlevels. Thus the invention provides a method or use for reducing TNF-αlevels in a human in need thereof comprising administering to the humanindividual the peptide of the invention in a pharmaceutically acceptablecarrier. In certain embodiments, the isolated peptide results in a 50%or 75% decrease in serum TNF-α levels when administered in an effectiveamount to a human subject compared to the levels before administrationof the isolated polypeptide. In other embodiments, the isolated peptidefurther comprises at least one other protein. The combination of the atleast two proteins can be referred to as a fusion protein. The otherprotein can be selected from an epitope tag and a half-life extender.The peptide can comprise both an epitope tag and a half-life extender.

We also provides a composition comprising an isolated peptide consistingessentially of or consisting of the amino acid sequenceX1-Z1-F-N-X2-P-F-X3-Z2-X4-Z3-X5 (SEQ ID NO: 3), wherein

X1 is V or L;

X2 is K or R;

X3 is V, L M, or Nle;

X4 is M, Nle, I or V;

X5 is K or Q;

Z1 is any amino acid;

Z2 is a sequence of any two amino acids;

Z3 is a sequence any five amino acids; and

wherein the isolated peptide causes a 75% decrease in serum TNF-α levelswhen administered in an effective amount to a human subject compared tothe amount of TNF-α levels before administering the peptide.

In certain embodiments, the isolated peptide comprises the amino acidsequence X1-Z1-F-N-X2-P-F-X3-Z2-X4-Z3-X5 (SEQ ID NO: 3), wherein X1, X2,X3, X4, X5, Z1, Z2, and Z3 are defined as above and wherein the peptideconsists of, at most, 35, 22 or 21 amino acid residues.

In certain aspects, the peptides of any of the embodiments describedherein and throughout the specification, also comprise at least oneother protein. The combination of these at least two proteins can bereferred to as a fusion protein. Specifically the other protein can beselected from an epitope tag and a half-life extender. In some aspectsof all the embodiments of the invention, the isolated peptide cancomprise both an epitope tag and a half-life extender.

The disclosure also provides an isolated peptide consisting essentiallyof or consisting of the amino acid sequence RFNRPFLR (SEQ ID NO: 4) andRFNKPFLR (SEQ ID NO: 5). In certain embodiments, the isolated peptidecauses a 50% or 75% decrease in serum TNF-α levels compared to theamount of TNF-α levels before administering the peptide whenadministered in an effective amount to a human subject.

In other aspects of all the embodiments of the invention, the isolatedpeptide is linked another protein. The combination of these proteins canbe referred to as a fusion protein. Specifically the other protein canbe selected an epitope tag and a half-life extender.

In some aspects of all the embodiments of the invention, the isolatedpeptide consists of, at most, 100, 35, 22, 21 or 9 additional aminoacids.

In other embodiments, the isolated peptide consists essentially of, orconsists of the amino acid sequence of Z1-RFNRPFLR-Z2 (SEQ ID NO: 6) andZ1-RFNKPFLR-Z2 (SEQ ID NO: 7), wherein Z1 and Z2 are independently 1, 2,3, 4, 5, 6, 6, 7, 8, 9, 10 or between 1 and 3, between 1 and 5, between1 and 6, between 1 and 7, between 1 and 8, between 1 and 9, or between 1and 10 basic amino acids.

In some embodiments, the isolated peptide consists essentially of orconsists of the amino acid sequence of RRRFNRPFLRRR (SEQ ID NO: 8) andRRRFNKPFLRRR (SEQ ID NO: 9).

The disclosure also provides a composition comprising an isolatedpeptide consisting essentially of or consisting of the amino acidsequence of FNRPFL (SEQ ID NO: 10) and FNKPFL (SEQ ID NO: 11).

The disclosure also provide a composition comprising an isolated orsynthesized peptide consisting essentially of or consisting of any oneor a combination of the following peptides: SP40; SP43; SP46; and SP49as set forth in Table B, and their use in methods for 1) treating adisease associated with a cytokine storm, 2) reducing an infarct size,and/or 3) treating acute myocardial infarction (AMI).

In some embodiments of any of the aspects described herein, the diseaseassociated with a cytokine storm is selected from the group consistingof acute myocardial infarction (AMI); gout; stroke; heart surgerycomplications; traumatic brain injury; acute respiratory distresssyndrome (ARDS), sepsis, Ebola, avian influenza, smallpox, and systemicinflammatory response syndrome (SIRS). In some embodiments of any of theaspects described herein, the human subject in need of a reduction ininfarct size is a subject in need of treatment for a disease selectedfrom the group consisting of: acute myocardial infarction (AMI);ischemia; stroke; traumatic brain injury; and toxic shock.

In one embodiment, the disclosure also provides a method of decreasingserum TNF-α compared to the amount of TNF-α levels before administeringthe peptide to a subject comprising administering to the subject aneffective amount of any one of the isolated peptides as defined above todecrease the serum TNF-α levels by at least 50%. In one embodiment,serum TNF-α levels are decreased by 75% compared to the amount of TNF-αlevels before administering the peptide. In other embodiments, thesubject is a mammal. In some aspects of all the embodiments of theinvention, the mammal is a human.

In some aspects of all the embodiments of the invention, the human hasbeen diagnosed with a disease associated with a cytokine storm, AMI, oran infarct prior to administering the peptide. In some aspects of allthe embodiments of the invention, the human has been diagnosed withacute myocardial infarction (AMI); gout, stroke, heart surgerycomplications, traumatic brain injury, acute respiratory distresssyndrome (ARDS), sepsis, Ebola, avian influenza, smallpox, systemicinflammatory response syndrome (SIRS), ischemia, and/or toxic shockprior to administering the peptide.

In some aspects, the human has not been subjected to prior treatmentwith alpha antitrypsin, such as alpha-1-antitrypsin treatment before thetreatment with the peptides of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a table showing the sequences and homology of SerpinC-terminal peptides (SEQ ID NOs: 18-24, respectively, in order ofappearance).

FIG. 2 is a table showing peptides derived from truncations (SEQ ID NOs:25-32, 1, 33-34, 1, 33, 38-51, 10, and 8, respectively, in order ofappearance).

FIGS. 3A-3D depict graphs and images demonstrating that AAT reducesinfarct size in experimental acute myocardial infarct (AMI) in themouse. FIG. 3A depicts a graph of the area at risk in the le. ventricle.FIG. 3B depicts a graph of infarct size (% of le. ventricle). CDD.Masson's trichrome stain of a midventricular heart section from arepresentative control mouse treated with albumin (FIG. 3C) or AAT (FIG.3D) 7 days after AMI. The infarct scar is evident in blue. Alb=albumin.

FIGS. 4A-4C depicts graphs demonstrating the efficacy of SP163M inreducing myocardial infarct size (FIG. 4A) reducing plasma levels ofcardiac specific troponin I, a biomarker of myocardial necrosis (FIG.4B), and, was also significantly reduced by SP16 treatment (FIG. 4B),and preserving the left ventricular systolic function, measured as leftventricular fractional shortening using transthoracic -echocardiography(FIG. 4C).

FIGS. 5A and 5B are graphs demonstrating that SP163M given with 30′ ofreperfusion significantly reduced infarct size (FIG. 5A) andpreservation of LV fractional shortening (FIG. 5B).

FIGS. 6A and 6B are graphs demonstrating that SP163M given with 30′ ofreperfusion reduces infarct size (FIG. 6A) and preserves LV fractionalshortening (FIG. 6B) in a dose-dependent fashion.

FIG. 7 is a graph demonstrating that SP163M does not affect heartcontractility. Depicted are measurements of cardiac systolic function atrest (left panel) or after isoproterenol challenge (contractile reserve)(right panel).

FIG. 8 depicts images and echocardiograms demonstrating that SP163Mreduces infarct size and effectively treats AMI.

FIG. 9 depicts a proposed model of the cytoprotective mechanism ofaction of SP16 and SP163M.

FIG. 10 depicts graphs of flow cytometry demonstrating that SP16increases expression of LRP1 on Raw264. 7 murine macrophages in adose-dependent manner.

FIG. 11 depicts a schematic of the onset and resolution of the acuteinflammatory response in acute myocardial infarction.

FIG. 12A and 12B depict SP16 binding to LRP1. When SERPINs bind theproteases inactivating them, a conformational change occurs by which ashort peptide containing a unique motif (5-11 amino acids) is exposed.FIG. 12A provides a schematic (modified from Joslin Get al. J Biol Chem1991 and Lillis A et al. Physiol Rev 2008) to illustrate that SP16contains AAT's pentapeptide, FVFLM (amino acid residues 7-11 of SEQ IDNO:1), which is responsible for binding to LRP1. FIG. 12B providesevidence that SP163M, and not SP34, binds to LRP1 in vitro.

FIG. 13A and 13B provide evidence that SP16 is an LRP1 agonist. SP163Minhibits NF-kB signaling induced by LPS (FIG. 13A) or Gp96 (FIG. 13B) inTHP1-XBlue™-MD2-CD14 cells, and that treatment with LRP1 blockingantibody or RAP, a co-factor leading to LRP1 downregulation, limitsSP16-related inhibition.

FIG. 14A and 14B demonstrate that LRP1 mediates SP16-inducedcardioprotection. Mice were pretreated with a LRP1 blocking antibody totest whether LRP1 mediated the cardioprotective signal of SP163M andAAT. Treatment with the blocking antibody eliminated the protectiveeffects of both SP163M and plasma derived AAT. N=5-8 per group.

DETAILED DESCRIPTION

The present disclosure describes a novel and unexpected use for isolatedpeptides in the field of disease treatment and prevention where thepeptides provide cytoprotective effects. Specifically, the inventiondescribes novel methods for treatment of diseases, where cytoprotectionis an essential component of disease treatment and/or prevention ofworsening of the condition, including e.g., acute myocardial infarction(AMI); gout; ischemia; stroke; traumatic brain injury; toxic shock, andheart surgery complications using isolated and/or synthesized peptideshaving unexpected cytoprotective properties.

Serine protease inhibitors (Serpins) represent a large (>1000) family ofprotease inhibitors, present in all branches of life and involved in amultitude of physiological processes. In mammals, such as humans,Serpins are important for homeostasis and although a certain level ofpromiscuity exists, each Serpin has a cognate serine protease(s). Forexample, alpha-1-antitrypsin (AAT) and alpha-1-antichymotrypsin (ACT)inhibit inflammatory proteases such as elastase, whereas antithrombininhibits thrombin and plays a role in coagulation.

A number of specific AAT mutations are manifested in human disease,including COPD, thrombosis and Serpinopathies (cirrhosis and dementia).Currently, a small number of human serum-derived AAT formulations areapproved by the FDA for treatment of COPD. In this therapeutic approach,AAT functions as a protease inhibitor similar to endogenous AAT.

AAT is the archetypical Serpin and shares tertiary structure with otherSerpins. Serpins have a ˜20 amino acid (aa) exposed loop, called thereactive center loop (RCL), which serves as bait for the cognateproteases. Once the protease binds the RCL, it becomes trapped,partially unfolded and destined for degradation. The cleavage of the RCLat its P1-P1′ site drives the process of protease inactivation andresults in the release of a small C-terminal peptide from the Serpinmolecule.

We have previously described Serpin peptide fragments and derivativesthereof that were determined to have anti-inflammatory properties (U.S.Patent No. 8,975,224).

Data presented herein, from an AMI model, demonstrate that AAT-derivedpeptides, e.g. SP16, not only have anti-inflammatory properties but alsodemonstrate surprising cytoprotective and infarct reducing activity. Thecytoprotective and infarct-reducing activities was not previously knownand provide significant and unexpected advantages for treatment ofcertain diseases with the peptides described herein. This isdemonstrated, for example, by the fact that SP16 is surprisinglyefficacious in treating AMI as compared to compounds with onlyanti-inflammatory activity (e.g. an IL-1 antagonist), providingactivities not achievable with mere anti-inflammatories, resulting insignificantly improved therapeutic effects.

Without wishing to be bound by theory, it is contemplated that SP16mediates the cardiac protection described herein by interfering withinflammatory cell death (pyroptosis) caused by ischemia and reperfusion.Specifically, SP16 limits acute ischemia-reperfusion injury, limits theacute inflammatory response in the heart, reduces infarct size, preventsadverse cardiac remodeling, and prevents heart failure. A proposed modelfor SP16's activity is depicted in FIG. 9.

This discovery leads us to provide methods of treating diseasesassociated with cytokine storms and/or reducing infarct size, e.g.treatment of AMI, by administering molecules from the C-terminalpeptides of Serpin molecules. The molecules are to be administered in anamount effective to reduce the size of an infarct or to reduce thecytodestructive effects of cytokine storms, such as ischemia reperfusioninjury.

We have discovered that SP16 (SEQ ID NO: 1) and SP163M (SEQ ID NO: 57),which is derived from human alpha-antitrypsin not only exhibitsanti-inflammatory and immune-modulatory properties similar to those ofthe parent protein, alpha-1-antitrypsin, but also acts as acytoprotective agent. SP16 appears to be a fist-in-class peptide masterswitch for treatment of autoimmune, inflammatory and metabolic diseasesand in addition, has a surprising effect as a cytoprotective agent.Without wishing to be bound by a theory, the peptides of the inventioncan provide a good safety profile, based on the good safety profile ofthe parent protein, alpha-1-antitrypsin. However, the peptides of theinvention are far easier and thus less expensive to produce as they aremuch smaller than the parent protein.

We have previously shown that C-terminal peptides that result from aSerpin molecule's cleavage by one of its cognate serine proteases have abiologic function that is distinct from that of the protease inhibitorfunction of the parent, complete Serpin molecule. For example, theC-terminal peptides from AAT, antichymotrypsin and Kallistatin havevarying degrees of anti-inflammatory effects. Rased on our currentresearch and surprising results, we now submit that therapeutic uses forthese peptides can be expanded from anti-inflammatory uses tocytoprotective uses. The novel function allows treatments aimed atpreventing, e.g., ischemia

The previously known functions of Serpins are related to inhibiting thefunction of serine protease enzymes. A few Serpins inhibit other typesof proteins, and several do not have an inhibitory function.

Serpins are a large family (>1000) of Serine Protease Inhibitors thatare structurally similar but functionally diverse. They are involved ina multitude of physiological processes and are critical for homeostasisin mammals. Genetic mutations in individual Serpins are manifested indifferent human diseases, including COPD, thrombosis and emphysema.

Each serpin with an inhibitory role is responsible for blocking theactivity of one or more proteins. Serpins bind to their target proteinsto prevent them from completing any further reactions. Upon binding to atarget, an irreversible change in the structure of a serpin proteinoccurs. Certain cells recognize when a Serpin is bound to its target andclear these attached proteins from the bloodstream.

Alpha-1-antitrypsin (AAT) is the prototypical Serpin. PROLASTIN®(Talecris), ZEMAIRA® (Aventis Behring) and ARALAST® (Baxter) are humanserum-derived AAT formulations approved by the FDA for treatment ofCOPD. AAT is currently in clinical trials for treatment of new onsettype I diabetes, graft vs. host disease and cystic fibrosis.

Researchers have identified at least 37 different serpin genes inhumans. Based on our research, we submit that isolated and synthesizedC-terminal fragments of the serpins proteins provide a novel source ofmolecules with anti-inflammatory, cytoprotective, and infarct reductiveactivity. Thus, we submit that the C-terminal fragments of at least theSerpins listed in Table A are useful in the methods described herein.

TABLE A Approved Symbol Approved Name Synonyms SERPINA1 serpin peptidaseinhibitor, clade A AAT, A1A, PI1, alpha-1- (alpha-1 antiproteinase,antitrypsin), antitrypsin, A1AT, alpha1AT member 1 SERPINA2 serpinpeptidase inhibitor, clade A ATR, ARGS (alpha-1 antiproteinase,antitrypsin), member 2 SERPINA3 serpin peptidase inhibitor, clade A ACT,alpha-1-antichymotrypsin (alpha-1 antiproteinase, antitrypsin), member 3SERPINA4 serpin peptidase inhibitor, clade A KST, KAL, KLST, kallistatin(alpha-1 antiproteinase, antitrypsin), member 4 SERPINA5 serpinpeptidase inhibitor, clade A PAI3, PROCI (alpha-1 antiproteinase,antitrypsin), member 5 SERPINA6 serpin peptidase inhibitor, clade A(alpha-1 antiproteinase, antitrypsin), member 6 SERPINA7 serpinpeptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin),member 7 AGT angiotensinogen (serpin peptidase inhibitor, clade A,member 8) SERPINA9 serpin peptidase inhibitor, clade A CENTERIN,SERPINA11b, (alpha-1 antiproteinase, antitrypsin), GCET1 member 9SERPINA10 serpin peptidase inhibitor, clade A PZI, ZPI (alpha-1antiproteinase, antitrypsin), member 10 SURPINA11 serpin peptidaseinhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 11SERPINA12 serpin peptidase inhibitor, clade A OL-64, Vaspin (alpha-1antiproteinase, antitrypsin), member 12 SERPINA13 serpin peptidaseinhibitor, clade A UNQ6121 (alpha-1 antiproteinase, antitrypsin), member13 (pseudogene) SERPINB1 serpin peptidase inhibitor, clade B EI, PI2,anti-elastase (ovalbumin), member 1 SERPINB2 serpin peptidase inhibitor,clade B HsT1201 (ovalbumin), member 2 SERPINB3 serpin peptidaseinhibitor, clade B T4-A, HsT1196 (ovalbumin), member 3 SERPINB4 serpinpeptidase inhibitor, clade B PI11, LEUPIN, SCCA-2, (ovalbumin), member 4SCCA1 SERPINB5 serpin peptidase inhibitor, clade B maspin (ovalbumin),member 5 SERPINB6 serpin peptidase inhibitor, clade B PTI, CAP(ovalbumin), member 6 SERPINB7 serpin peptidase inhibitor, clade BMEGSIN (ovalbumin), member 7 SERPINB8 serpin peptidase inhibitor, cladeB CAP2 (ovalbumin), member 8 SERPINB9 serpin peptidase inhibitor, cladeB CAP3 (ovalbumin), member 9 SERPINB10 serpin peptidase inhibitor, cladeB bomapin (ovalbumin), member 10 SERPINB11 serpin peptidase inhibitor,clade B EPIPIN (ovalbumin), member 11 (gene/pseudogene) SERPINB12 serpinpeptidase inhibitor, clade B YUKOPIN (ovalbumin), member 12 SERPINB13serpin peptidase inhibitor, clade B HUR7, hurpin, headpin (ovalbumin),member 13 SERPINC1 serpin peptidase inhibitor, clade C ATIII, MGC22579(antithrombin), member 1 SERPIND1 serpin peptidase inhibitor, clade DHC-II, HLS2, HC2, D22S673 (heparin cofactor), member 1 SERPINE1 serpinpeptidase inhibitor, clade E PAI (nexin, plasminogen activator inhibitortype 1), member 1 SERPINE2 serpin peptidase inhibitor, clade E PN1, GDN,PNI, nexin (nexin, plasminogen activator inhibitor type 1), member 2SERPINE3 serpin peptidase inhibitor, clade E (nexin, plasminogenactivator inhibitor type 1), member 3 SERPINF1 serpin peptidaseinhibitor, clade F EPC-1, PIG35 (alpha-2 antiplasmin, pigment epitheliumderived factor), member 1 SERPINF2 serpin peptidase inhibitor, clade FAPI, ALPHA-2-PI, A2AP, (alpha-2 antiplasmin, pigment AAP epitheliumderived factor), member 7 SERPING1 serpin peptidase inhibitor, clade GC1IN, C1-INH, HAE1, HAE2 (C1 inhibitor), member 1 SERPINH1 serpinpeptidase inhibitor, clade H HSP47, collagen (heat shockprotein 47),member 1, (collagen binding protein 1) SERPINI1 serpin peptidaseinhibitor, clade I neuroserpin (neuroserpin), member 1 SERPINI2 serpinpeptidase inhibitor, clade I PANCPIN, TSA2004, MEPI, (pancpin), member 2pancpin

The peptides described in U.S. Pat. No. 8,975,224 are specificallydefined short isolated or synthesized C-terminal peptides based onSerpins and variants and derivatives thereof with surprisingly effectiveanti-inflammatory properties and with much more useful size fortherapeutic applications compared to the native Serpin proteins. Theisolated peptides are shown in FIGS. 1-2. FIG. 1 shows the amino acidsequences of the C-terminal fragments of a variety of Serpins. Eachpeptide is marked with a SEQ ID NO: in column 2, immediately to the leftof the peptide. FIG. 2 shows truncations of the C-terminal fragmentsshown in FIG. 1, as well as variants and derivatives thereof. Again,each peptide is marked with a SEQ ID NO: in column 2, immediately nextto the peptide.

An alanine screen demonstrated that isolated or synthesized or modifiedSP16 peptides shown in Table B below are particularly effective inreducing TNF-alpha levels in a mouse model for inflammation.Specifically, in these particular fragments, the three most N-terminaland the two most C-terminal amino acids appear to play a role in theanti-inflammatory properties of the peptides as replacement of themappeared to reduce the capacity of the peptides to reduce TNF-alphalevels in a LPS challenged mouse model of sepsis (see, e.g.,PCT/US13/20498; which is incorporated by reference herein in itsentirety). Accordingly, in some aspects of all embodiments of theinvention the peptides are selected from SP40, SP43, SP46, and SP49 thepeptide sequences of which are set forth in Table B.

Table B shows peptides named SP16; SP40; SP43; SP46; and SP49 providedparticularly good anti-inflammatory effect when administered to a mousemodel of sepsis. SP16, SP163M, SP37, SP40, and/or SP51 bind LRP1 and arecontemplated to be cytoprotective as described herein. In someembodiments, SP16, SP163M, SP37, SP40, and/or SP51 can be used in themethods described herein.

TABLE B Peptide Amino Acid Sequence SP16V K F N K P F V F L M I E Q N T K (SEQ ID NO: 1) SP37

(SEQ ID NO: 12) SP40

(SEQ ID NO: 13) SP43

(SEQ ID NO: 14) SP46

(SEQ ID NO: 15) SP49

(SEQ ID NO: 16) SP51

(SEQ ID NO: 17)

According to some embodiments and aspects of the invention, method ofproviding cytoprotective treatment comprise administering to a subject,such as a human subject, an effective cytoprotection or infarctsize-reducing amount of any of isolated peptides consisting of orconsisting essentially of sequences set forth in SEQ ID NOs: 8, 10,19-34, and 38-49 can be used in the methods described herein. Any ofpeptides consisting of or consisting essentially of sequences set forthSEQ ID NOs: 8, 10, 19-34, and 38-49 can also be used to reduce TNF-α ina subject. In certain embodiments, the amount of TNF-α in the serum isreduced by up to 50% or more or 75% or more compared to the amount ofthe same in the serum prior to administering the peptide.

Table C below presents additional exemplary peptides that were used toreduce TNF-alpha levels in mice subjected to an LPS challenge. Also thepeptides with capacity to reduce TNF-alpha levels as described inPCT/US13/20498; which is incorporated by reference herein in itsentirety, are contemplated for the compositions, pharmaceuticalcompositions and methods of use and treatment of inflammatory conditionsin the present invention.

TABLE C(SEQ ID NOS 18-32, 1, and 33-34, respectively, in order of appearance)SP1 Human AATC36S I P P E V K F N K P F VF L M I E Q N T K S P L F M G K V V N P T Q KHuman SP2 KALLISTATINS A Q T N R H I L R F N R P F L V V I F S T S T Q S V L F L G K V V D P T K P(C39) Human SP3 AntichymotrypsinS A L V E T R T I V R F N R P F L M I I V P T D T Q N I F F M S K V T N P K Q A(C40) SP4 Rat Serpina3MS G R P P M I V W F N R P F L I A V S H T H G Q T I L F M A K V I N P V G A(C38) SP5 Rat Serpina3KK S L P Q T I P L L N F N R P F M L V I T D D N G Q S V F F M G K V T N P M(C38) SP6 Human Hybrid 1 S A Q V E T I V R F N R P F L V I I V S T N T QSP7 Human-Rat S I P P Q M I V W F N R P F L I A I S H T H T Q Hybrid 1SP8 Human AAT C36 S I P P E V K F N K P F V F L M I E Q N T K Human SP9KALLISTATIN S A Q T N R H I L R F N R P F L V V I F S T S T Q(C39) Human SP10 AntichymotrypsinS A L V E T R T I V R F N R P F L M I I V P T D T Q (C40) SP11Rat SerPina3K K S L P Q T I P L L N F N R P F M L V I T D D N G Q (C38)SP12 Human AAT C36 I P P E V K F N K P F V F L M I E Q N T K Human SP13KALLISTATIN N R H I L R F N R P F L V V I F S T S T Q (C39) Human SP14Antichymotrypsin T R T I V R F N R P F L M I I V P T D T Q (C40) SP15Rat Serpina3K T I P L L N F N R P F M L V I T D D N G Q (C38) SP16C36 Core V K F N K P F V F L M I E Q N T K sequence, long SP17 C36 CoreV K F N K P F V F L M sequence, short SP18 Human AAT C36S I P P E V K A A A A A A F L M I E Q N T K SP1 (SEQ ID NO: 18); SP2(SEQ ID NO: 19); SP3 (SEQ ID NO: 20); SP4 (SEQ ID NO: 21); SP5 (SEQ IDNO: 22); SP6 (SEQ ID NO: 23); SP7 (SEQ ID NO: 24); SP8 (SEQ ID NO: 25);SP9 (SEQ ID NO: 26); SP10 (SEQ ID NO: 27); SP11 (SEQ ID NO: 28); SP12(SEQ ID NO: 29); SP13 (SEQ ID NO: 30); SP14 (SEQ ID NO: 31); SP15 (SEQID NO: 32); SP16 (SEQ ID NO: 1); SP163M (SEQ ID No: 57) SP17 (SEQ ID NO:33); SP18 (SEQ ID NO: 34).

The phrase “consisting essentially of” is herein meant to define thescope of the peptides to the specified material amino acids, and to onlyinclude additional amino acids or changes that do not materially affectthe claimed invention's basic and novel characteristics, namely, theanti-inflammatory capacity of the short isolated or synthesizedpeptides. The definition specifically excludes peptides that have asequence of a complete Serpin protein, and the definition alsospecifically excludes peptide sequences that are equal to or longer than37 amino acids of any naturally occurring Serpin protein.

Without wishing to be bound by a theory, we have also identified theimportant amino acids that provide the core, and the possiblemodifications, for, e.g., anti-inflammatory peptides as manufacturedherein. The isolated peptides encompassed by the formulae set forthbelow are also provided and they can be used to reduce inflammation,treat cytokine storms, enhance cell survival, and/or reduce infarctsize.

Human AAT, antichymotrypsin, and kallistatin have been known to containelements with anti-inflammatory properties. However, these elements havenot been previously identified. We have now established a new family ofhuman Serpin-derived peptides with potent anti-inflammatory effectusing, e.g., a mouse endotoxemia model (LPS induced endotoxemia). Rasedon the efficacy of the peptides in the mouse inflammation model, thepeptide size, and the safety profile of the parent protein, theAAT-based peptides, the peptides, such as SP16, and fragments andderivatives thereof provide a novel and improved molecule to treatinflammation in humans.

Formula I provides a composition comprising a peptide comprising,consisting essentially of or consisting of the amino acid sequenceX1-Z1-F-N-R-P-F-X2-Z2-X3-Z3-Q (SEQ ID NO:35) andX1-Z1-F-N-K-P-F-X2-Z2-X3-Z3 (SEQ ID NO: 2) wherein

X1 is V or L;

X2 is V, L or M or Nle;

X3 is M or Nle, I or V;

Z1 is any amino acid;

Z2 is a sequence of any two amino acids; and

Z3 is a sequence any five amino acids, wherein the peptide comprises 37or fewer amino acids.

In certain aspects of all the embodiments, the isolated peptide causes a50% or 75% decrease in serum TNF-α levels compared to the serum level asmeasured prior to administering the isolated peptide, when administeredin an effective amount to a human subject. In some aspects of all theembodiments of the invention, the peptide further comprises a fusionprotein. The fusion protein can be selected from an epitope tag and ahalf-life extender or a combination thereof.

Formula II provides a composition comprising an isolated peptidecomprising, consisting essentially of or consisting of the amino acidsequence X1-Z1-F-N-X2-P-F-X3-Z2-X4-Z3-X5 (SEQ ID NO: 3), wherein

X1 is V or L;

X2 is K or R;

X3 is V, L M, or Nle;

X4 is M, or Nle, I or V;

X5 is K or Q;

Z1 is any amino acid;

Z2 is a sequence of any two amino acids;

Z3 is a sequence any five amino acids; and wherein the isolated peptidecauses a 75% decrease in serum TNF-α levels compared to the serum levelsprior to administering the isolated peptide when administered in aneffective amount to a human subject.

In certain embodiments, the peptide comprising the amino acid sequenceof X1-Z1-F-N-X2-P-F-X3-Z2-X4-Z3-X5 (SEQ ID NO: 3) as defined above,includes, at most, 35, 22 or 21 amino acid residues. In certain aspectsof all the embodiments of the invention, the peptide further comprises afusion protein. Specifically the fusion protein can be selected from anepitope tag and a half-life extender or a combination thereof.

In some aspects of all the methods and uses of the invention, thepeptide is SP16.

In some aspects of all the methods and uses of the invention, thepeptide can comprise the peptide of SEQ ID NO: 57. In some aspects ofall the methods and uses of the invention, the peptide can consistessentially of the peptide of SEQ ID NO: 57.

Therefore, the invention also provides methods and uses relating to anisolated peptide consisting of or consisting essentially of the aminoacid sequence RFNRPFLR (SEQ ID NO: 4) and RFNKPFLR (SEQ ID NO: 5), whichcan also be used for the treatment of inflammation. In certainembodiments, the peptide causes a 50% or 75% decrease in serum TNF-αlevels compared to the serum TNF-α levels prior to administering theisolated peptide when administered in an effective amount to a humansubject. In some aspects of all the embodiments of the invention, theisolated peptide further comprises a fusion protein. Specifically thefusion protein can be selected from an epitope tag and a half-lifeextender. In other embodiments, the isolated peptide comprises, at most,100, 35, 22, 21, 16 or 9 amino. In other embodiments, the isolatedpeptide comprises the amino acid sequence of Z1-RFNRPFLR-Z2 (SEQ ID NO:6), and Z1-RFNKPFLR-Z2 (SEQ ID NO: 7) wherein Z1 and Z2 areindependently between 1, 2, 3, 4, 5, 6, 6, 7, 8, 9, 10 or between 1 and3, between 1 and 5, between 1 and 6, between 1 and 7, between 1 and 8,between 1 and 9, or between 1 and 10 basic amino acids.

In some aspects of all the embodiments of the invention, the isolatedpeptide consists essentially of or consists of the amino acid sequenceof RRRFNRPFLRRR (SEQ ID NO: 8) and RRRFNKPFLRRR (SEQ ID NO: 9). Thedisclosure also provides a composition comprising a peptide consistingessentially of or consisting of the amino acid sequence of FNRPFL (SEQID NO: 10) and FNKPFL (SEQ ID NO: 11).

In certain embodiments the isolated peptide comprises 5 or moresequential amino acids from the amino acid sequence of FLMIEQNTK (SEQ IDNO: 36). These peptides can be used to reduce inflammation, enhance cellsurvival, treat cytokine storms, reduce infarct size and/or reduce TNF-αlevel in a subject. In certain embodiments, the amount of TNF-α level inthe serum is reduced compared to the amount of TNF-α in the serum priorto administering the isolated peptide.

In some aspects, the method of treatment further comprises analysis ofTNF-α serum levels prior to administering the isolated peptide and afteradministering the isolated peptide. If the TNF-α serum level isdecreased less than 30%, the step of administering can be repeated withthe same dose or with a larger dose of the peptide compared to the firstdose.

Fragments of any of the peptides described above can vary in size. Forexample, these fragments can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, or 37, amino acids in length.

The peptides described above can, in addition to treating diseasesassociated with cytokine storms and/or reducing infarct size, e.g.treatment of AMI, reduce inflammation. The peptides exertanti-inflammatory and immune-modulating effects, and additionally,directly or indirectly stimulate beta cell regeneration. In certainembodiments, these peptides reduce inflammation by reducing the activityor expression of TNF-α. The activity of TNF-α can be reduced by 10, 15,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99 or100%. The expression of TNF-α can be reduced 10, 15, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99 or 100%. Whenadministered therapeutically, the peptide composition typically furthercomprises a pharmaceutically acceptable solution or carrier.

The peptides described above can also be used to treat, prevent orimprove the symptoms of several pathologies. Accordingly, the disclosurefurther provides methods of treating a disease associated with acytokine storm or methods of reducing infract size, comprising the stepof administering any one of the peptides described herein or acombination thereof to a subject in need of treatment. In some aspects,the subject has not been treated with alpha-antitrypsin prior to thetreatment. In some aspects, the method comprises a step of assayingwhether the individual has increased serum TNF-α levels and if thesubject has increased serum TNF-α levels then administering the peptideto the subject, and if not, then not administering the peptide to thesubject.

The disclosure also enables methods of preventing development of adisease associated with a cytokine storm or an preventing an increase ininfarct size comprising the step of administering any one of thepeptides or a combination thereof to a subject in need of prevention ofa disease associated with a cytokine storm or prevention of an increasein infarct size. In some aspects, the subject has not been treated withalpha-antitrypsin prior to the treatment. In some aspects, the methodcomprises a step of assaying whether the individual has increased serumTNF-α levels and if the subject has increased serum TNF-α levels thenadministering the peptide to the subject, and if not, then notadministering the peptide to the subject. In some aspects of all theembodiments of the invention, the subject in need thereof has not beenpreviously treated with any of the peptides as described herein for anycondition.

In some aspects of all embodiments of the invention the primary purposeof the treatment is to treat or prevent undesired cell death and tissuedeterioration associated with deleterious after effects of acutemyocardial infarction (AMI); gout; stroke; heart surgery complications;traumatic brain injury. The effective dosage in such applications ismeasured by determining the effect on prevention or improvement ofischemia reperfusion injury. The dosage is not necessarily the same asin treatment of inflammation alone. Effectiveness can also be measuredby determining the level of cell viability and/or cell death, e.g., in aspecific organ or tissue.

For convenience, certain terms employed in the entire application(including the specification, examples, and appended claims) are definedthroughout the specification. Unless defined otherwise, all technicaland scientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs.

The term “wild type” refers to the naturally-occurring polynucleotidesequence encoding a protein, or a portion thereof, or protein sequence,or portion thereof, respectively, as it normally exists in vivo.

The term “mutant” refers to any change in the genetic material of anorganism, in particular a change (i.e., deletion, substitution,addition, or alteration) in a wild-type polynucleotide sequence or anychange in a wild-type protein sequence. Although it is often assumedthat a change in the genetic material results in a change of thefunction of the protein, the term—“mutant” refers to a change in thesequence of a wild-type protein regardless of whether that change altersthe function of the protein (e.g., increases, decreases, imparts anewfunction), or whether that change has no effect on the function of theprotein (e.g., the mutation or variation is silent). The term mutationis used interchangeably herein with polymorphism in this application.

The terms “polypeptide” and “protein” are used interchangeably to referto an isolated polymer of amino acid residues, and are not limited to aminimum length unless otherwise defined. Peptides, oligopeptides,dimers, multimers, and the like, are also composed of linearly arrangedamino acids linked by peptide bonds, and whether produced biologicallyand isolated from the natural environment, produced using recombinanttechnology, or produced synthetically typically using naturallyoccurring amino acids.

In some aspects, the polypeptide or protein is a “modified polypeptide”comprising non-naturally occurring amino acids. In some aspects, thepolypeptides comprise a combination of naturally occurring andnon-naturally occurring amino acids, and in some embodiments, thepeptides comprise only non-naturally occurring amino acids.

In some aspects of all the embodiments of the invention, the peptides ormodified peptides further comprise co-translational andpost-translational (C-terminal peptide cleavage) modifications, such as,for example, disulfide-bond formation, glycosylation, acetylation,phosphorylation, proteolytic cleavage (e.g., cleavage by furins ormetalloproteases), and the like to the extent that such modifications donot affect the anti-inflammatory properties of the isolated peptides ortheir capacity to improve glycemic control.

In some aspects of the invention, the polypeptide is altered. The term“altered polypeptide” refers to a peptide that includes alterations,such as deletions, additions, and substitutions (generally conservativein nature as would be known to a person in the art, such as alanines),to the native sequence, as long as the protein maintains the desiredactivity, i.e., it anti-inflammatory activity of capacity to improveglycemic control or reduce hyperglycemia. These modifications can bedeliberate, as through site-directed mutagenesis, or can be accidental,such as through mutations of artificial hosts, such as geneticallyengineered bacteria, yeast or mammalian cells, that produce theproteins, or errors due to PCR amplification or other recombinant DNAmethods. Polypeptides or proteins are composed of linearly arrangedamino acids linked by peptide bonds, but in contrast to peptides, have awell-defined conformation. Proteins, as opposed to peptides, generallyconsist of chains of 50 or more amino acids.

The term “peptide” as used herein typically refers to a sequence ofamino acids made up of a single chain of amino acids joined by peptidebonds. Generally, peptides contain at least two amino acid residues andare less than about 50 amino acids in length, unless otherwise defined.

“Modified peptide” may include the incorporation of non-natural aminoacids into the peptides of the invention, including synthetic non-nativeamino acids, substituted amino acids, or one or more D-amino acids intothe peptides (or other components of the composition, with exception forprotease recognition sequences) is desirable in certain situations.D-amino acid-containing peptides exhibit increased stability in vitro orin vivo compared to L-amino acid-containing forms. Thus, theconstruction of peptides incorporating D-amino acids can be particularlyuseful when greater in vivo or intracellular stability is desired orrequired. More specifically, D-peptides are resistant to endogenouspeptidases and proteases, thereby providing better oral trans-epithelialand transdermal delivery of linked drugs and conjugates, improvedbioavailability of membrane-permanent complexes (see below for furtherdiscussion), and prolonged intravascular and interstitial lifetimes whensuch properties are desirable. The use of D-isomer peptides can alsoenhance transdermal and oral trans-epithelial delivery of linked drugsand other cargo molecules. Additionally, D-peptides cannot be processedefficiently for major histocompatibility complex class II-restrictedpresentation to T helper cells, and are therefore less likely to inducehumoral immune responses in the whole organism. Peptide conjugates cantherefore be constructed using, for example, D-isomer forms of cellpenetrating peptide sequences, L-isomer forms of cleavage sites, andD-isomer forms of therapeutic peptides. Therefore, in some embodimentsthe peptides as disclosed comprise L and D amino acids, wherein no morethan 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 D-amino acids are included. Incertain aspects, the peptides comprise more than 10 D-amino acids, andin certain aspects all the amino acids of the peptides are D-aminoacids.

In yet a further aspect, the peptides or fragments or derivativesthereof can be “retro-inverso peptides.” A “retro-inverso peptide”refers to a peptide with a reversal of the direction of the peptide bondon at least one position, i.e., a reversal of the amino- andcarboxy-termini with respect to the side chain of the amino acid. Thus,a retro-inverso analogue has reversed termini and reversed direction ofpeptide bonds while approximately maintaining the topology of the sidechains as in the native peptide sequence. The retro-inverso peptide cancontain L-amino acids or D-amino acids, or a mixture of L-amino acidsand D-amino acids, up to all of the amino acids being the D-isomer.Partial retro-inverso peptide analogues are polypeptides in which onlypart of the sequence is reversed and replaced with enantiomeric aminoacid residues. Since the retro-inverted portion of such an analogue hasreversed amino and carboxyl termini, the amino acid residues flankingthe retro-inverted portion are replaced by side-chain-analogousα-substituted geminal-diaminomethanes and malonates, respectively.Retro-inverso forms of cell penetrating peptides have been found to workas efficiently in translocating across a membrane as the natural forms.Synthesis of retro-inverso peptide analogues are described in Bonelli,F. et al., Int J Pept Protein Res. 24(6):553-6 (1984); Verdini, A andViscomi, G. C, J. Chem. Soc. Perkin Trans. 1:697-701 (1985); and U.S.Pat. No. 6,261,569, which are incorporated herein in their entirety byreference. Processes for the solid-phase synthesis of partialretro-inverso peptide analogues have been described (EP 97994 B) whichis also incorporated herein in its entirety by reference.

The terms “homology”, “identity” and “similarity” refer to the degree ofsequence similarity between two peptides or between two optimallyaligned nucleic acid molecules. Homology and identity can each bedetermined by comparing a position in each sequence which can be alignedfor purposes of comparison. For example, it is based upon using standardhomology software in the default position, such as BLAST, version2.2.14. When an equivalent position in the compared sequences isoccupied by the same base or amino acid, then the molecules areidentical at that position; when the equivalent site occupied by similaramino acid residues (e.g., similar in steric and/or electronic naturesuch as, for example conservative amino acid substitutions), then themolecules can be referred to as homologous (similar) at that position.Expression as a percentage of homology/similarity or identity refers toa function of the number of similar or identical amino acids atpositions shared by the compared sequences, respectfully. A sequencewhich is “unrelated” or “non-homologous” shares less than 40% identity,though preferably less than 25% identity with the sequences as disclosedherein.

As used herein, the term “sequence identity” means that twopolynucleotide or amino acid sequences are identical (i.e., on anucleotide-by-nucleotide or residue-by-residue basis) over thecomparison window. The term “percentage of sequence identity” iscalculated by comparing two optimally aligned sequences over the windowof comparison, determining the number of positions at which theidentical nucleic acid base (e.g., A, T. C, G. U. or I) or residueoccurs in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the comparison window (i.e., the window size), andmultiplying the result by 100 to yield the percentage of sequenceidentity.

The term “substantial identity” as used herein denotes a characteristicof a polynucleotide or amino acid sequence, wherein the polynucleotideor amino acid comprises a sequence that has at least 85% sequenceidentity, preferably at least 90% to 95% sequence identity, more usuallyat least 99% sequence identity as compared to a reference sequence overa comparison window of at least 18 nucleotide (6 amino acid) positions,frequently over a window of at least 24-48 nucleotide (8-16 amino acid)positions, wherein the percentage of sequence identity is calculated bycomparing the reference sequence to the sequence which can includedeletions or additions which total 20 percent or less of the referencesequence over the comparison window. The reference sequence can be asubset of a larger sequence. The term “similarity”, when used todescribe a polypeptide, is determined by comparing the amino acidsequence and the conserved amino acid substitutes of one polypeptide tothe sequence of a second polypeptide.

As used herein, the terms “homologous” or “homologues” are usedinterchangeably, and when used to describe a polynucleotide orpolypeptide, indicates that two polynucleotides or polypeptides, ordesignated sequences thereof, when optimally aligned and compared, forexample using BLAST, version 2.2.14 with default parameters for analignment (see herein) are identical, with appropriate nucleotideinsertions or deletions or amino-acid insertions or deletions, in atleast 70% of the nucleotides, usually from about 75% to 99%, and morepreferably at least about 98 to 99% of the nucleotides. The term“homolog” or “homologous” as used herein also refers to homology withrespect to structure and/or function. With respect to sequence homology,sequences are homologs if they are at least 50%, at least 60 at least70%, at least 80%, at least 90%, at least 95% identical, at least 97%identical, or at least 99% identical. Determination of homologs of thegenes or peptides of the present invention can be easily ascertained bythe skilled artisan.

The term “substantially homologous” refers to sequences that are atleast 90%, at least 95% identical, at least 96%, identical at least 97%identical, at least 98% identical or at least 99% identical. Homologoussequences can be the same functional gene in different species.Determination of homologs of the genes or peptides of the presentinvention can be easily ascertained by the skilled artisan.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for comparison can be conducted, forexample, by the local homology algorithm of Smith and Waterman (Adv.Appl. Math. 2:482 (1981), which is incorporated by reference herein), bythe homology alignment algorithm of Needleman and Wunsch (J. MoI. Biol.48:443-53 (1970), which is incorporated by reference herein), by thesearch for similarity method of Pearson and Lipman (Proc. Natl. Acad.Sci. USA 85:2444-48 (1988), which is incorporated by reference herein),by computerized implementations of these algorithms (e.g., GAP, BESTFIT,FASTA, and TFASTA in the Wisconsin Genetics Software Package, GeneticsComputer Group, 575 Science Dr., Madison, Wis.), or by visualinspection. (See generally Ausubel et al. (eds.), Current Protocols inMolecular Biology, 4th ed., John Wiley and Sons, New York (1999)).

One example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments to show the percent sequence identity. It also plotsa tree or dendogram showing the clustering relationships used to createthe alignment. PILEUP uses a simplification of the progressive alignmentmethod of Feng and Doolittle (J. MoI. Evol. 25:351-60 (1987), which isincorporated by reference herein). The method used is similar to themethod described by Higgins and Sharp (Comput. Appl. Biosci. 5:151-53(1989), which is incorporated by reference herein). The program canalign up to 300 sequences, each of a maximum length of 5,000 nucleotidesor amino acids. The multiple alignment procedure begins with thepairwise alignment of the two most similar sequences, producing acluster of two aligned sequences. This cluster is then aligned to thenext most related sequence or cluster of aligned sequences. Two clustersof sequences are aligned by a simple extension of the pairwise alignmentof two individual sequences. The final alignment is achieved by a seriesof progressive, pairwise alignments. The program is run by designatingspecific sequences and their amino acid or nucleotide coordinates forregions of sequence comparison and by designating the programparameters. For example, a reference sequence can be compared to othertest sequences to determine the percent sequence identity relationshipusing the following parameters: default gap weight (3.00), default gaplength weight (0.10), and weighted end gaps.

Another example of an algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described by Altschul et al. (J. MoI. Biol. 215:403-410 (1990), whichis incorporated by reference herein). (See also Zhang et al., NucleicAcid Res. 26:3986-90 (1998); Altschul et al., Nucleic Acid Res.25:3389-402 (1997), which are incorporated by reference herein).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information internet web site. Thisalgorithm involves first identifying high scoring sequence pairs (HSPs)by identifying short words of length W in the query sequence, whicheither match or satisfy some positive-valued threshold score T whenaligned with a word of the same length in a database sequence. T isreferred to as the neighborhood word score threshold (Altschul et al.(1990), supra). These initial neighborhood word hits act as seeds forinitiating searches to find longer HSPs containing them. The word hitsare then extended in both directions along each sequence for as far asthe cumulative alignment score can be increased. Extension of the wordhits in each direction is halted when: the cumulative alignment scorefalls off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. The BLAST algorithm parameters W, T, and Xdetermine the sensitivity and speed of the alignment. The BLAST programuses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix(see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-9(1992), which is incorporated by reference herein) alignments (B) of 50,expectation (E) of 10, M=5, N=−4, and a comparison of both strands.

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin and Altschul, Proc. Natl. Acad. Sci.USA 90:5873-77 (1993), which is incorporated by reference herein). Onemeasure of similarity provided by the BLAST algorithm is the smallestsum probability (P(N)), which provides an indication of the probabilityby which a match between two nucleotide or amino acid sequences wouldoccur by chance. For example, an amino acid sequence is consideredsimilar to a reference amino acid sequence if the smallest sumprobability in a comparison of the test amino acid to the referenceamino acid is less than about 0.1, more typically less than about 0.01,and most typically less than about 0.001.

The term “variant” as used herein refers to a peptide or nucleic acidthat differs from the polypeptide or nucleic acid by one or more aminoacid or nucleic acid deletions, additions, substitutions or side-chainmodifications, yet retains one or more specific functions or biologicalactivities of the naturally occurring molecule. Amino acid substitutionsinclude alterations in which an amino acid is replaced with a differentnaturally-occurring or a non-conventional amino acid residue. Suchsubstitutions may be classified as “conservative”, in which case anamino acid residue contained in a polypeptide is replaced with anothernaturally occurring amino acid of similar character either in relationto polarity, side chain functionality or size. Such conservativesubstitutions are well known in the art. Substitutions encompassed bythe present invention may also be “non-conservative”, in which an aminoacid residue which is present in a peptide is substituted with an aminoacid having different properties, such as naturally-occurring amino acidfrom a different group (e.g., substituting a charged or hydrophobicamino; acid with alanine), or alternatively, in which anaturally-occurring amino acid is substituted with anon-conventionalamino acid. In some embodiments amino acid substitutions areconservative. Also encompassed within the term variant when used withreference to a polynucleotide or polypeptide, refers to a polynucleotideor polypeptide that can vary in primary, secondary, or tertiarystructure, as compared to a reference polynucleotide or polypeptide,respectively (e.g., as compared to a wild-type polynucleotide orpolypeptide).

Variants can also be synthetic, recombinant, or chemically modifiedpolynucleotides or polypeptides isolated or generated using methods wellknown in the art. Variants can include conservative or non-conservativeamino acid changes, as described below. Polynucleotide changes canresult in amino acid substitutions, additions, deletions, fusions andtruncations in the polypeptide encoded by the reference sequence.Variants can also include insertions, deletions or substitutions ofamino acids, including insertions and substitutions of amino acids andother molecules) that do not normally occur in the peptide sequence thatis the basis of the variant, for example but not limited to insertion ofornithine which do not normally occur in human proteins. The term“conservative substitution,” when describing a polypeptide, refers to achange in the amino acid composition of the polypeptide that does notsubstantially alter the polypeptide's activity. For example, aconservative substitution refers to substituting an amino acid residuefor a different amino acid residue that has similar chemical properties.Conservative amino acid substitutions include replacement of a leucinewith an isoleucine or valine, an aspartate with a glutamate, or athreonine with a serine.

“Conservative amino acid substitutions” result from replacing one aminoacid with another having similar structural and/or chemical properties,such as the replacement of a leucine with an isoleucine or valine, anaspartate with a glutamate, or a threonine with a serine. Thus, a“conservative substitution” of a particular amino acid sequence refersto substitution of those amino acids that are not critical forpolypeptide activity or substitution of amino acids with other aminoacids having similar properties (e.g., acidic, basic, positively ornegatively charged, polar or non-polar, etc.) such that the substitutionof even critical amino acids does not reduce the activity of thepeptide, (i.e. the ability of the peptide to penetrate the blood brainbarrier (BBB)). Conservative substitution tables providing functionallysimilar amino acids are well known in the art. For example, thefollowing six groups each contain amino acids that are conservativesubstitutions for one another: 1) Alanine (A), Serine (S), Threonine(T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N),Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine(L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W). (See also Creighton, Proteins, W. H. Freeman and Company(1984), incorporated by reference in its entirety.) In some embodiments,individual substitutions, deletions or additions that alter, add ordelete a single amino acid or a small percentage of amino acids can alsobe considered “conservative substitutions” if the change does not reducethe activity of the peptide. Insertions or deletions are typically inthe range of about 1 to 5 amino acids. The choice of conservative aminoacids may be selected based on the location of the amino acid to besubstituted in the peptide, for example if the amino acid is on theexterior of the peptide and expose to solvents, or on the interior andnot exposed to solvents.

In alternative embodiments, one can select the amino acid which willsubstitute an existing amino acid based on the location of the existingamino acid, i.e. its exposure to solvents (i.e. if the amino acid isexposed to solvents or is present on the outer surface of the peptide orpolypeptide as compared to internally localized amino acids not exposedto solvents). Selection of such conservative amino acid substitutionsare well known in the art, for example as disclosed in Dordo et al, J.MoI Biol, 1999, 217, 721-739 and Taylor et al, J. Theor. Biol.119(1986);205-218 and S. French and B. Robson, J. MoI. Evol.19(1983)171. Accordingly, one can select conservative amino acidsubstitutions suitable for amino acids on the exterior of a protein orpeptide (i.e. amino acids exposed to a solvent), for example, but notlimited to, the following substitutions can be used: substitution of Ywith F, T with S or K, P with A, E with D or Q, N with D or G, R with K,G with N or A, T with S or K, D with N or E, I with L or V, F with Y, Swith T or A, R with K, G with N or A, K with R, A with S, K or P.

In alternative embodiments, one can also select conservative amino acidsubstitutions encompassed suitable for amino acids on the interior of aprotein or peptide, for example one can use suitable conservativesubstitutions for amino acids is on the interior of a protein or peptide(i.e. the amino acids are not exposed to a solvent), for example but notlimited to, one can use the following conservative substitutions: whereY is substituted with F, T with A or S, I with L or V, W with Y, M withL, N with D, G with A, T with A or S, D with N, I with L or V, F with Yor L, S with A or T and A with S, G, Tor V. In some embodiments,non-conservative amino acid substitutions are also encompassed withinthe term of variants.

The term “derivative” as used herein refers to peptides which have beenchemically modified, for example but not limited to by techniques suchas ubiquitination, labeling, pegylation (derivatization withpolyethylene glycol), lipidation, glycosylation, or addition of othermolecules. A molecule also a “derivative” of another molecule when itcontains additional chemical moieties not normally a part of themolecule. Such moieties can improve the molecule's solubility,absorption, biological half-life, etc. The moieties can alternativelydecrease the toxicity of the molecule, eliminate or attenuate anyundesirable side effect of the molecule, etc. Moieties capable ofmediating such effects are disclosed in Remington's PharmaceuticalSciences, 18th edition, A R. Gennaro, Ed., MackPubl., Easton, Pa.(1990), incorporated herein, by reference, in its entirety.

Thus, in certain aspects of all the embodiments of the invention, thepeptides of the invention comprise peptide derivatives, such aspegylated peptides.

The term “functional” when used in conjunction with “derivative” or“variant” refers to a peptide of the invention which possesses abiological activity (either functional or structural) that issubstantially similar to a biological activity of the entity or moleculeit is a functional derivative or functional variant thereof, i.e.,anti-inflammatory activity in the context of the peptides describedherein. The term functional derivative is intended to include thefragments, analogues or chemical derivatives of a molecule.

The term “insertions” or “deletions” are typically in the range of about1 to 5 amino acids. The variation allowed can be experimentallydetermined by producing the peptide synthetically while systematicallymaking insertions, deletions, or substitutions of nucleotides in thesequence using recombinant DNA techniques.

The term “substitution” when referring to a peptide, refers to a changein an amino acid for a different entity, for example another amino acidor amino-acid moiety. Substitutions can be conservative ornon-conservative substitutions.

An “analog” of a molecule such as a peptide refers to a molecule similarin function to either the entire molecule or to a fragment thereof. Theterm “analog” is also intended to include allelic species and inducedvariants. Analogs typically differ from naturally occurring peptides atone or a few positions, often by virtue of conservative substitutions.Analogs typically exhibit at least 80 or 90% sequence identity withnatural peptides. Some analogs also include unnatural amino acids ormodifications of N or C terminal amino acids. Examples of unnaturalamino acids are, for example but not limited to; disubstituted aminoacids, N-alkyl amino acids, lactic acid, 4-hydroxyproline,γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine,O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine,5-hydroxylysine, σ-N-methylarginine. Fragments and analogs can bescreened for prophylactic or therapeutic efficacy in transgenic animalmodels as described below.

By “covalently bonded” is meant joined either directly or indirectly(e.g., through a linker) by a covalent chemical bond. In some aspects ofall the embodiments of the invention, the fusion peptides are covalentlybonded.

The term “fusion protein” as used herein refers to a recombinant proteinof two or more proteins. Fusion proteins can be produced, for example,by a nucleic acid sequence encoding one protein is joined to the nucleicacid encoding another protein such that they constitute a singleopen-reading frame that can be translated in the cells into a singlepolypeptide harboring all the intended proteins. The order ofarrangement of the proteins can vary. Fusion proteins can include anepitope tag or a half-life extender. Epitope tags include biotin, FLAGtag, c-myc, hemaglutinin, His6 (SEQ ID NO: 37), digoxigenin, FITC, Cy3,Cy5, green fluorescent protein, V5 epitope tags, GST, β-galactosidase,AU1, AUS, and avidin. Half-life extenders include Fc domain and serumalbumin.

The terms “subject” and “individual” and “patient” are usedinterchangeably herein, and refer to an animal, for example a human ornon-human animal (e.g., a mammal) , to whom treatment, includingprophylactic treatment, with a pharmaceutical composition as disclosedherein, is provided. The term “subject” as used herein refers to humanand non-human animals. The term “non-human animals” includes allvertebrates, e.g., mammals, such as non-human primates, (particularlyhigher primates), sheep, dogs, rodents (e.g. mouse or rat), guinea pigs,goats, pigs, cats, rabbits, cows, and non-mammals such as chickens,amphibians, reptiles etc. In one embodiment, the subject is human. Inanother embodiment, the subject is an experimental animal or animalsubstitute as a disease model. Non-human mammals include mammals such asnon-human primates, (particularly higher primates), sheep, dogs, rodents(e.g. mouse or rat), guinea pigs, goats, pigs, cats, rabbits and cows.In some aspects, the non-human animal is a companion animal such as adog or a cat.

“Treating” a disease or condition in a subject or “treating” a patienthaving a disease or condition refers to subjecting the individual to apharmaceutical treatment, e.g., the administration of a drug, such thatat least one symptom of the disease or condition is decreased orstabilized. Typically, when the peptide is administered therapeuticallyas a treatment, it is administered to a subject who presents with one ormore symptoms of, e.g., AMI.

The term “prevention” is used in connection of prevention of symptoms orslowing down of symptom development from the time of asymptomatic state.Typically, when the peptide is administered preventively, it isadministered to a subject who does not present imminent symptoms of acondition described herein, e.g. AMI. Typically, the subject is at riskof developing an infarct or a disease associated with a cytokine storm,such as AMI, due to the family history, laboratory results, genetictesting or life-style. The peptide may also be administered within 10-30minutes or 10-60 minutes, or within 10 minutes to 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24hours, up to 2, 3, 4, 5, days after onset of symptoms of, e.g., AMI orstroke or after diagnosis of the same to prevent worsening of symptoms.The peptide may also be administered within 10-30 minutes or 10-60minutes, or within 10 minutes to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, up to 2, 3, 4,5, days after injury and/or index event to prevent worsening ofsymptoms. In some embodiments of any of the aspects described herein,the peptide may be administered within 10-30 minutes, 30-60 minutes, or10-60 minutes, after injury and/or index event.

By “specifically binds” or “specific binding” is meant a compound orantibody that recognizes and binds a desired polypeptide but that doesnot substantially recognize and bind other molecules in a sample, forexample, a biological sample, which naturally includes a polypeptide ofthe invention. Specific binding can be characterized by a dissociationconstant of at least about 1×10−6 M or smaller. In other embodiments,the dissociation constant is at least about 1×10−7 M, 1×10−8 M , or1×10−9 M. Methods for determining whether two molecules specificallybind are well known in the art and include, for example, equilibriumdialysis, surface plasmon resonance, and the like.

By “isolated” it is meant that the polypeptide has been separated fromany natural environment, such as a body fluid, e.g., blood, andseparated from the components that naturally accompany the peptide.

By isolated and “substantially pure” is meant a polypeptide that hasbeen separated and purified to at least some degree from the componentsthat naturally accompany it. Typically, a polypeptide is substantiallypure when it is at least about 60%, or at least about 70%, at leastabout 80%, at least about 90%, at least about 95%, or even at leastabout 99%, by weight, free from the proteins and naturally-occurringorganic molecules with which it is naturally associated. For example, asubstantially pure polypeptide may be obtained by extraction from anatural source, by expression of a recombinant nucleic acid in a cellthat does not normally express that protein, or by chemical synthesis.

By a “decrease” or “inhibition” used in the context of the level of, forexample TNF-alpha levels refers to reduction of the amount of protein inthe biological sample, such as blood or tissue sample, a cell, a cellextract, or a cell supernatant. For example, such a decrease may be dueto reduced RNA stability, transcription, or translation, increasedprotein degradation, or RNA interference. Preferably, this decrease isat least about 5%, at least about 10%, at least about 25%, at leastabout 50%, at least about 75%, at least about 80%, or even at leastabout 90% compared to a reference value.

The term “reference value” in the context of the claims and theapplication refers typically to an abnormally high TNF-alpha level foundin an individual affected with or suffering from inflammation. Thereference value is typically the amount of TNF-alpha in the individualprior to administering of the peptide of the invention. In some aspectsof all the embodiments concerning glycemic control, the term “referencevalue” refers to the numeric values used in measuring glycemic controlin a subject. There are a number of tests which can be used to determineif, e.g., a human subject is affected with pre-diabetes. Such testsinclude, e.g., the AlC test, fasting plasma glucose test (FPG), and theoral glucose tolerance test (OGTT).

By an “increase” in the expression or activity of a gene or protein ismeant a positive change in protein or nucleic acid level or activity ina cell, a cell extract, or a cell supernatant. For example, such anincrease may be due to increased RNA stability, transcription, ortranslation, or decreased protein degradation. Preferably, this increaseis at least 5%, at least about 10%, at least about 25%, at least about50%, at least about 75%, at least about 80%, at least about 100%, atleast about 200%, or even about 500% or more over the level ofexpression or activity under control conditions.

The term “recombinant” as used herein to describe a nucleic acidmolecule, means a polynucleotide of genomic, cDNA, viral, semisynthetic,and/or synthetic origin, which, by virtue of its origin or manipulation,is not associated with all or a portion of the polynucleotide with whichit is associated in nature. The term recombinant as used with respect toa protein or polypeptide, means a polypeptide produced by expression ofa recombinant polynucleotide. The term recombinant as used with respectto a host cell means a host cell into which a recombinant polynucleotidehas been introduced. Recombinant is also used herein to refer to, withreference to material (e.g., a cell, a nucleic acid, a protein, or avector) that the material has been modified by the introduction of aheterologous material (e.g., a cell, a nucleic acid, a protein, or avector).

The term “vectors” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked; a plasmidis a species of the genus encompassed by “vector”. The term “vector”typically refers to a nucleic acid sequence containing an origin ofreplication and other entities necessary for replication and/ormaintenance in a host cell. Vectors capable of directing the expressionof genes and/or nucleic acid sequence to which they are operativelylinked are referred to herein as “expression vectors”. In general,expression vectors of utility are often in the form of “plasmids” whichrefer to circular double stranded DNA loops which, in their vector formare not bound to the chromosome, and typically comprise entities forstable or transient expression or the encoded DNA Other expressionvectors can be used in the methods as disclosed herein for example, butare not limited to, plasmids, episomes, bacterial artificialchromosomes, yeast artificial chromosomes, bacteriophages or viralvectors, and such vectors can integrate into the host's genome orreplicate autonomously in the particular cell. A vector can be a DNA orRNA vector. Other forms of expression vectors known by those skilled inthe art which serve the equivalent functions can also be used, forexample self-replicating extrachromosomal vectors or vectors whichintegrates into a host genome. Preferred vectors are those capable ofautonomous replication and/or expression of nucleic acids to which theyare linked. Vectors capable of directing the expression of genes towhich they are operatively linked are referred to herein as “expressionvectors”.

The term “viral vectors” refers to the use of viruses, orvirus-associated vectors as carriers of a nucleic acid construct into acell. Constructs may be integrated and packaged into non-replicating,defective viral genomes like Adenovirus, Adeno-associated virus (AAV),or Herpes simplex virus (HSV) or others, including reteroviral andlentiviral vectors, for infection or transduction into cells. The vectormay or may not be incorporated into the cell's genome. The constructsmay include viral sequences for transfection, if desired. Alternatively,the construct may be incorporated into vectors capable of episomalreplication, e.g. EPV and EBV vectors.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., at least one) of the grammatical object of the article.By way of example, “an element” means one element or more than oneelement. Other than in the operating examples, or where otherwiseindicated, all numbers expressing quantities of ingredients or reactionconditions used herein should be understood as modified in all instancesby the term “about.” The term “about” when used in connection withpercentages can mean+1%. The present invention is further explained indetail by the following examples, but the scope of the invention shouldnot be limited thereto.

It should be understood that this invention is not limited to theparticular methodology, protocols, and reagents, etc., described hereinand as such can vary. The terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention, which is defined solely by the claims.Other features and advantages of the invention will be apparent from thefollowing Detailed Description, the drawings, and the claims. Treatmentmethods of the invention

One aspect of the present invention relates to the use of peptidesdescribed herein and mutants, variants, analogs or derivatives thereof.Specifically, these methods relate to administering any one of thepeptides as described herein or their pharmaceutically acceptablemodifications in a pharmaceutically acceptable carrier to a subject,e.g., a mammal in need thereof, e.g., a human, i.e., a subject having adisease associated with a cytokine storm; a subject in need of areduction of infarct size; and/or a subject in need of treatment of AMI.Clinical descriptions of these diseases and conditions are well known.In some aspects the human is first diagnosed as having one or moresymptom of the disease before administering one or more of the peptidesof the invention. In some embodiments, the human has not previously beenadministered AAT as a treatment for the symptoms.

As used herein, a disease “associated with a cytokine storm” is adisease in which a cytokine storm is a cause of, symptomatic of, or aresult of the pathogenesis of the disease. Non-limiting examples ofdiseases associated with a cytokine storm can include acute myocardialinfarction (AMI); gout; stroke; heart surgery complications; and/ortraumatic brain injury.

A subject in need of a reduction in infarct size can be a subject withan infarct. A subject in need of a reduction in infarct size can be asubject having or diagnosed as having, e.g., acute myocardial infarction(AMI); ischemia; stroke; traumatic brain injury; and/or toxic shocks.

As used herein, “acute myocardial infarction” or “AMI” refers to areduction or cessation of blood flow to the heart, which can result indamage to the heart tissue from a variety of causes.

In some embodiments of any of the aspects described herein, the subjectdoes not have a condition, does not have symptoms of the condition,and/or is not diagnosed as having the condition selected from the groupconsisting of type II diabetes, lupus, graft versus host disease,uveitis, eczema, psoriasis, cystic fibrosis, rheumatoid arthritis, acuteradiation syndrome, burn patients, inflammatory bowel disease, type Idiabetes, and hyperglycemia.

For example, we have shown in established preclinical mouse models forhuman diseases including AMI that, for example, the SP16 peptidesignificantly improves the symptoms. For example, SP16 was shown toreduce infarct size.

We have also provided evidence that, e.g., the SP16 peptide can besafely administered using well-established preclinical safety studies.For example, we have also shown that SP16 does not affect heartcontractility in the AMI model. Additionally, it is has been shown usingFastPatch assay that, for example, the SP16 peptide does not impact hERGactivity and we also were not able to identify any hits on humanreceptor panning study (GenSEP Explorer) for the SP16 peptide.

Therefore, in one aspect we provide methods for treatment of a subjecthaving a disease associated with a cytokine storm; a subject in need ofa reduction of infarct size; and/or a subject in need of treatment ofAMI, comprising administering to a human subject in need thereof acomposition comprising at least one of the peptides of the invention. Insome aspects, the peptide comprises SP16.

We have shown that the peptides of the invention, e.g., SP16 are tolllike receptor-2 agonists. Accordingly, without wishing to be bound by atheory, the peptides, such as SP16, act as an anti-inflammatory drug bypromoting an anti-inflammatory cytokine profile. Also, without wishingto be bound by a theory, the peptides, such as SP16, also can act asimmune modulators by inducing expansion of tolerogenic and protectiveT-regulatory cells (T-regs). Further, without wishing to be bound by atheory, the peptides, such as SP16, also can down-regulate autoimmuneresponses without inducing general immunological suppression therebyproviding a superior treatment for autoimmune diseases compared to mostof the currently available treatments which generally suppress theimmune system exposing the treated individuals to a risk of infectionswhile treated with the general immunosuppressants.

As the peptides are derived from AAT, and in view of our results in vivoand in vitro models, it is reasonable to expect most of the AAT'stherapeutic effects to apply also to the peptides of the invention, suchas SP16. Specifically, AAT has been shown to modulate T-cellproliferation and NF-kappa-B activation; impair NK target cellinteraction; inhibit serine proteases activation of epithelial cellEGFR/TLR-4 signaling; be involved in TNF-alpha-induced gene expressionand apoptosis or endothelial cells; prevent red blood cell haemolysis byE. coli, decrease circulating eosinophil cell count; inhibit neutrophilchemotaxis, NADHP oxidase and ANCA signaling; inhibit monocyte andmacrophage cytokine release and regulation of CD14 expression, andinhibit mast cell histamine release; and modulate B-cell proliferationand cytokine production. In some aspects, the peptide is SP16, which maycomprise one or more modifications typically performed to enhancepeptide bioavailability and/or shelf life, such as pegylation and thelike.

We also performed a peptide optimization assay using an alanine scanwith the TLR-2 assay. Data was obtained using an experiment with anengineered TLR-2 indicator cell line (HEK-BLUE™ mTLR2, Invivogen). Cellswere incubated with the 20 μg/ml of the indicated peptides for 24 hours.Upon TLR2 activation, the cells secrete alkaline phosphatase, which canbe assayed. The assay was done in triplicate and averages are plotted.Peptide SP34 is a scrambled peptide control (Yellow), and PAM (Pam3CSK4;Red) is a positive control. See, e.g., PCT/US13/20498; which isincorporated by reference herein in its entirety.

The following table provides the results from an assay for SP16'spharmacokinetic profile.

PK parameters SP16, IV (5 mg/kg) C0 (μg/mL) 2.5 AUG to Last (μg-hr/mL)0.9 t½ (hr) 1.9 Total CL (mL/hr) 140 Total CL (mL/min/kg) 9.7 Last Timepoint 8.0 MRTINF (hr) 1.1 V (mL) 374 Vss (mL) 149

In performing the assay, three normal rats were injected intravenouslywith 5 mg/kg SP16 and the plasma concentration of SP16 established at 8time points following the injection. For each timepoint, SP16 levelswere determined by LC/MS/MS and the values used to calculate the Cmax(2.5 ug/ml) and T1/2 (1.9 hours). The assay was executed by Apredica,Boston, Mass. Accordingly, we determined that SP16 has a half-life of1.9 hours in normal rats.

The SP16 safety profile also included hERG data. The hERG FastPatchassay showed that SP16 does not inhibit hERG at up to 25 μM doses. Thisdata predicts that SP16 will not have cardiac safety issues in humans.The study was executed by Apredica, Boston, Mass.

Test conc IC50 value Client ID (μM) (μM) comment SP16 0.008-25 >25 Noconcentration-dependent inhibition observed. Quinidine  0.01-10 1.8positive control Mean % Activity 0 0.008 0.04 0.2 1 5 25 Customer Id μMμM μM μM μM μM μM Comments SP16 100 89 95 95 78 88 89 Noconcentration-dependent inhibition observed

In addition, we also performed a profile using human receptor panning.The GenSEP Explorer panel contains 111 in vitro assay targets carefullyselected to assess drug/chemical biological activities. Assay categoriesinclude GPCRs, Voltage-Gated Ion Channels, Ligand-Gated Ion Channels,Neurotransmitter transporters, Nuclear Receptors and Steroids as well asa diverse set of biochemical targets including phosphodiesterases,kinases and other relevant enzymes. The study was executed by CaliperLifeSciences, and the results are summarized in the table below. Itappears that SP16 has no effect on 111 human receptors, indicating thatSP16 has an excellent human safety profile.

Number of Receptors Percent Receptor Class Tested inhibitionNeurotransmitter Related 47 Insignificant Steroids 4 Insignificant IonChannels 8 Insignificant Growth factors & hormones 2 InsignificantSecond Messengers 3 Insignificant Brain/gut peptides 15 InsignificantEnzymes 20 Insignificant Enzymes, Kinases 11 Insignificant Cell-Based,Functional 1 Insignificant

In view of the safety profile of SP16, we can reasonably extrapolatethat the other peptide fragments provided herein would also be safe foradministering to human.

In one embodiment, the methods of treatment described herein, furthercomprise selection or diagnosis of a subject having any of theabove-described conditions, e.g., one arising from inflammation prior toadministering a peptide as disclosed herein or a mutant, variant, analogor derivative thereof, to thereby treat the condition or dysfunction,such as inflammation. Such selection is performed by the skilledpractitioner by a number of available methods, for instance, assessmentof symptoms which are described herein. For example, AMI can bediagnosed by means of, e.g., an electrocardiogram, a chest radiograph,or the level of troponins. For example, one can assess the amount ofTNF-alpha in the subject to determine the amount of inflammation presentin the subject.

In some aspects of all the embodiments, one may use C-reactive proteinas a marker for inflammation or treatment efficacy. C-reactive protein(CRP) is used to detect inflammation if there is a high suspicion oftissue injury or infection somewhere in the body. CRP serves as ageneral marker for infection and inflammation and can be used toevaluate an individual for an acute or chronic inflammatory condition. Ahigh or increasing amount of CRP in the blood suggests the presence ofinflammation. In individuals suspected of having a serious bacterialinfection, a high CRP suggests the presence of one. In people withchronic inflammatory conditions, high levels of CRP suggest a flare-upor that treatment has not been effective. Normal concentration inhealthy human serum is usually lower than 10 mg/L, slightly increasingwith aging. Higher levels are found in late pregnant women, mildinflammation and viral infections (10-40 mg/L), active inflammation,bacterial infection (40-200 mg/L), severe bacterial infections and burns(>200 mg/L). In some aspects the term “reference value” refers to themeasurements of CRP when CRP is used as a diagnostic test forinflammation.

Successful or effective treatment is evidenced by amelioration of one ormore symptoms of the condition or dysfunction as discussed herein.Administering a peptide as disclosed herein or a mutant, variant, analogor derivative thereof in a subject in need thereof is expected toprevent or retard the development of the conditions and physicaldysfunctions described herein (e.g., those arising from an infarct, acytokine storm, inflammation or auto-immune tissue destruction or to acondition ameliorated by stimulation of expansion of beta cell mass inan individual with diabetes). The term “prevention” is used to refer toa situation wherein a subject does not yet have the specific conditionbeing prevented, meaning that it has not manifested in any appreciableform. Prevention encompasses prevention or slowing of onset and/orseverity of a symptom, (including where the subject already has one ormore symptoms of another condition). Prevention is performed generallyin a subject who is at risk for development of a condition or physicaldysfunction. Such subjects are said to be in need of prevention. Forexample, reduction in the TNF-alpha levels compared to the levels priorto administering the peptides of the invention, would be evidence ofsuccessful treatment.

In one embodiment, the methods of prevention described herein, furthercomprise selection of such a subject at risk for a condition, e.g.,those arising from an infarct, a cytokine storm, inflammation orauto-immune tissue destruction or physical dysfunction as describedherein, prior to administering a peptide or a mutant, variant, analog orderivative thereof, in the subject, to thereby prevent the condition ordysfunction. Such selection is performed by the skilled practitioner bya number of available methods. For instance, assessment of risk factorsor diagnosis of a disease which is known to cause the condition ordysfunction, or treatment or therapy known to cause the condition ordysfunction. Subjects which have a disease or injury or a relevantfamily history which is known to contribute to the condition aregenerally considered to be at increased risk.

As used herein, the terms “treat” or “treatment” or “treating” refers totherapeutic treatment measures, wherein the object is to prevent or slowthe development of the disease, such as reducing at least one effect orsymptom of a condition, disease or disorder associated withinflammation. Treatment is generally “effective” if one or more symptomsare improved or clinical markers, such as troponins, TNF-alpha, CRP,blood glucose and/or HbAlc, levels are within normal values or closer tothe normal reference values than abnormal values reflecting inflammationor poor glycemic control, depending on the condition, as that term isdefined herein. Alternatively, treatment is “effective” if theprogression of a disease is slowed down, exhibition of a symptom or amarker for a disease is reduced. That is, “treatment” includes theimprovement of symptoms or markers, slowing of progress or slowing ofworsening of at least one symptom that would be expected in absence oftreatment. Beneficial or desired clinical results include, but are notlimited to, alleviation of one or more symptom(s), diminishment ofextent of disease, stabilized (i.e., not worsening) state of disease,delay or slowing of disease progression, amelioration or palliation ofthe disease state. “Treatment” can also mean prolonging survival ascompared to expected survival if not receiving treatment. Those in needof treatment include patients with one or more symptoms of an infarct,cytokine storm, or inflammation, such as symptoms associated with AMI.

TNF-alpha levels can be assessed, for example, using any number ofreadily available commercial ELISA kits.

In some aspects, the invention relates to methods of preventing acytokine storm, an infarct, and/or AMI by administering the peptides asdescribed to an individual not yet presenting symptoms of a cytokinestorm, an infarct, and/or AMI. For example, the peptides can beadministered to an individual at high risk of developing AMI, but notyet having AMI to assist in slowing down the development or preventingthe development of AMI.

The term “effective amount” as used herein refers to the amount of apharmaceutical composition comprising one or more peptides as disclosedherein or a mutant, variant, analog or derivative thereof, to decreaseat least one or more symptom of the disease or disorder, and relates toa sufficient amount of pharmacological composition to provide thedesired effect. The phrase “therapeutically effective amount” as usedherein means a sufficient amount of the composition to treat a disorder,at a reasonable benefit/risk ratio applicable to any medical treatment.The term “therapeutically effective amount” therefore refers to anamount of the composition as disclosed herein that is sufficient toeffect a therapeutically or prophylactically reduction in a symptom orclinical marker associated with increased levels of inflammation,infract, or cytokine storm when administered to a typical subject whohas, e.g., AMI. Typically reduction of more than 20% of a diseasemarker, such as an inflammatory marker, e.g., TNF-alpha, is indicativeof effective treatment. In some instances, reduction of more than 50% ormore than 75% from the amount of TNF-alpha levels in the individualprior to administering the peptides of the invention is indicative ofeffective treatment.

A therapeutically or prophylactically significant reduction in a symptomis, e.g. at least about 10%, at least about 20%, at least about 30%, atleast about 40%, at least about 50%, at least about 60%, at least about70%, at least about 80%, at least about 90%, at least about 100%, atleast about 125%, at least about 150% or more in a measured parameter ascompared to a control or non-treated subject or the state of the subjectprior to administering the peptide. Measured or measurable parametersinclude clinically detectable markers of disease, for example, elevatedor depressed levels of a biological marker, such as TNF-alpha, as wellas parameters related to a clinically accepted scale of symptoms ormarkers for infarct, cytokine storms, and inflammation. It will beunderstood, however, that the total daily usage of the compositions andformulations as disclosed herein will be decided by the attendingphysician within the scope of sound medical judgment. The exact amountrequired will vary depending on factors such as the type of diseasebeing treated, gender, age, and weight of the subject.

With reference to the treatment a subject having a disease associatedwith a cytokine storm; a subject in need of a reduction of infarct size;and/or a subject in need of treatment of AMI, the term “therapeuticallyeffective amount” refers to the amount that is safe and sufficient todelay the development of one or more symptom and results in decrease inthe amount of a disease marker, e.g., TNF-α or CRP concentrations. Theeffective amount for the treatment of a disease depends on the type ofdisease, the species being treated, the age and general condition of thesubject, the mode of administration and so forth. Thus, it is notpossible to specify the exact “effective amount.” However, for any givencase, an appropriate “effective amount” can be determined by one ofordinary skill in the art using only routine experimentation. Theefficacy of treatment can be judged by an ordinarily skilledpractitioner, for example, efficacy can be assessed in known animalmodels of inflammation (e.g. LPS model), AMI (e.g. an isoproterenolmodel), auto-immune tissue destruction (e.g. CAIA model) or diabetes(e.g. db/db mouse model). When using an experimental animal model,efficacy of treatment is evidenced when a reduction in a symptom of acytokine storm or infarct is shown versus untreated animals.

In some embodiments of any of the aspects described herein, the subjectdoes not have, does not have symptoms of, or is not diagnosed as havinga condition selected from the group consisting of type II diabetes,lupus, graft versus host disease, uveitis, eczema, psoriasis, cysticfibrosis, rheumatoid arthritis, acute radiation syndrome, burn patients,inflammatory bowel disease, type I diabetes, and hyperglycemia. In someembodiments of any of the aspects described herein, the subject does nothave increased TNF-α levels. In some embodiments of any of the aspectsdescribed herein, the subject is not in need of a reduction in TNF-αlevels.

As used herein, the terms “administering,” and “introducing” are usedinterchangeably herein and refer to the placement of the therapeuticagents such as one or more peptides as disclosed herein or a mutant,variant, analog or derivative thereof into a subject by a method orroute which results in delivering of such agent(s) at a desired site.The compounds can be administered by any appropriate route which resultsin an effective treatment in the subject.

The one or more peptides as disclosed herein or a mutant, variant,analog or derivative thereof may be administered by any route known inthe art or described herein, for example, oral, parenteral (e.g.,intravenously or intramuscularly), intraperitoneal, rectal, cutaneous,nasal, vaginal, inhalant, skin (patch), or ocular. The one or morepeptides as disclosed herein or a mutant, variant, analog or derivativethereof may be administered in any dose or dosing regimen. One can alsouse pumps, like the ones used for insulin administration. In someembodiments, the one or more peptides can be administered orally. Asdepicted in FIG. 16 of U.S. Pat. No. 8,975,224; which is incorporated byreference herein in its entirety), oral administration of SP16 wasdemonstrated to be efficacious, e.g. compared to intraperitonealinjection of SP16 and dexamethas one.

Dosage

With respect to the therapeutic methods of the invention, it is notintended that the administration of the one or more peptides asdisclosed herein or a mutant, variant, analog or derivative thereof andbe limited to a particular mode of administration, dosage, or frequencyof dosing; the present invention contemplates all modes ofadministration, including intramuscular, intravenous, intraperitoneal,intravesicular, intraarticular, intralesional, subcutaneous, or anyother route sufficient to provide a dose adequate to treat the, e.g.,inflammation, infarct, or cytokine storm-related disorder. Thetherapeutic may be administered to the patient in a single dose or inmultiple doses. When multiple doses are administered, the doses may beseparated from one another by, for example, one hour, three hours, sixhours, eight hours, one day, two days, one week, two weeks, or onemonth. For example, the therapeutic may be administered for, e.g., 2, 3,4, 5, 6, 7, 8, 10, 15, 20, or more weeks. It is to be understood that,for any particular subject, specific dosage regimes should be adjustedover time according to the individual need and the professional judgmentof the person administering or supervising the administration of thecompositions. For example, the dosage of the therapeutic can beincreased if the lower dose does not provide sufficient therapeuticactivity.

While the attending physician ultimately will decide the appropriateamount and dosage regimen, therapeutically effective amounts of the oneor more peptides as disclosed herein or a mutant, variant, analog orderivative thereof may be provided at a dose of 0.0001, 0.01, 0.01 0.1,1, 5, 10, 25, 50, 100, 500, or 1,000 mg/kg or μg/kg. Effective doses maybe extrapolated from dose-response curves derived from in vitro oranimal model test bioassays or systems.

Dosages for a particular patient or subject can be determined by one ofordinary skill in the art using conventional considerations, (e.g. bymeans of an appropriate, conventional pharmacological protocol). Aphysician may, for example, prescribe a relatively low dose at first,subsequently increasing the dose until an appropriate response isobtained. The dose administered to a patient is sufficient to effect abeneficial therapeutic response in the patient over time, or, e.g., toreduce symptoms, or other appropriate activity, depending on theapplication. The dose is determined by the efficacy of the particularformulation, and the activity, stability or serum half-life of the oneor more peptides as disclosed herein or a mutant, variant, analog orderivative thereof and the condition of the patient, as well as the bodyweight or surface area of the patient to be treated. The size of thedose is also determined by the existence, nature, and extent of anyadverse side-effects that accompany the administration of a particularvector, formulation, or the like in a particular subject. Therapeuticcompositions comprising one or more peptides as disclosed herein or amutant, variant, analog or derivative thereof are optionally tested inone or more appropriate in vitro and/or in vivo animal models ofdisease, such as models of inflammation or diabetes, to confirmefficacy, tissue metabolism, and to estimate dosages, according tomethods well known in the art. In particular, dosages can be initiallydetermined by activity, stability or other suitable measures oftreatment vs. non-treatment (e.g., comparison of treated vs. untreatedcells or animal models), in a relevant assay. Formulations areadministered at a rate determined by the LD50 of the relevantformulation, and/or observation of any side-effects of one or morepeptides as disclosed herein or a mutant, variant, analog or derivativethereof. Administration can be accomplished via single or divided doses.

In determining the effective amount of one or more peptides as disclosedherein or a mutant, variant, analog or derivative thereof to beadministered in the treatment or prophylaxis of disease the physicianevaluates circulating plasma levels, formulation toxicities, andprogression of the disease.

The efficacy and toxicity of the compound can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., ED50 (the dose is effective in 50% of the population) and LD50(the dose is lethal to 50% of the population). The dose ratio of toxicto therapeutic effects is the therapeutic index, and it can be expressedas the ratio, LD50/ED50. Pharmaceutical compositions which exhibit largetherapeutic indices are preferred.

These compounds may be administered to humans and other animals fortherapy by any suitable route of administration that works for smallpeptides, including orally, nasally, as by, for example, a spray,rectally, intravaginally, parenterally, intracisternally and topically,as by powders, ointments or drops, including buccally and sublingually.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient which is effective to achieve the desiredtherapeutic response for a particular subject, composition, and mode ofadministration, without being toxic to the subject.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compound employed, the age, sex, weight, condition, generalhealth and prior medical history of the patient being treated, and likefactors well known in the medical arts.

Formulation of Pharmaceutical Compositions—“Pharmaceutically AcceptableCarriers”

The administration of one or more peptides as disclosed herein or amutant, variant, analog or derivative thereof may be by any suitablemeans that results in a concentration of the protein that treats thedisorder. The compound may be contained in any appropriate amount in anysuitable carrier substance, and is generally present in an amount of1-95% by weight of the total weight of the composition. The compositionmay be provided in a dosage form that is suitable for the oral,parenteral (e.g., intravenously or intramuscularly), intraperitoneal,rectal, cutaneous, nasal, vaginal, inhalant, skin (patch), or ocularadministration route. Thus, the composition may be in the form of, e.g.,tablets, capsules, pills, powders, granulates, suspensions, emulsions,solutions, gels including hydrogels, pastes, ointments, creams,plasters, drenches, osmotic delivery devices, suppositories, enemas,injectables, implants, sprays, or aerosols. The pharmaceuticalcompositions may be formulated according to conventional pharmaceuticalpractice (see, e.g., Remington: The Science and Practice of Pharmacy,20th edition, 2000, ed. A. R. Gennaro, Lippincott Williams & Wilkins,Philadelphia, and Encyclopedia of Pharmaceutical Technology, eds. J.Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York,incorporated, herein, by reference in its entirety).

Pharmaceutical compositions according to the invention may be formulatedto release the active compound immediately upon administration or at anypredetermined time or time period after administration. The latter typesof compositions are generally known as controlled release formulations,which include (i) formulations that create substantially constantconcentrations of the agent(s) of the invention within the body over anextended period of time; (ii) formulations that after a predeterminedlag time create substantially constant concentrations of the agent(s) ofthe invention within the body over an extended period of time; (iii)formulations that sustain the agent(s) action during a predeterminedtime period by maintaining a relatively constant, effective level of theagent(s) in the body with concomitant minimization of undesirable sideeffects associated with fluctuations in the plasma level of the agent(s)(sawtooth kinetic pattern); (iv) formulations that localize action ofagent(s), e.g., spatial placement of a controlled release compositionadjacent to or in the diseased tissue or organ; (v) formulations thatachieve convenience of dosing, e.g., administering the composition onceper week or once every two weeks; and (vi) formulations that target theaction of the agent(s) by using carriers or chemical derivatives todeliver the therapeutic to a particular target cell type. Administrationof the protein in the form of a controlled release formulation isespecially preferred for compounds having a narrow absorption window inthe gastrointestinal tract or a relatively short biological half-life.

Any of a number of strategies can be pursued in order to obtaincontrolled release in which the rate of release outweighs the rate ofmetabolism of the compound in question. In one example, controlledrelease is obtained by appropriate selection of various formulationparameters and ingredients, including, e.g., various types of controlledrelease compositions and coatings. Thus, the protein is formulated withappropriate excipients into a pharmaceutical composition that, uponadministration, releases the protein in a controlled manner. Examplesinclude single or multiple unit tablet or capsule compositions, oilsolutions, suspensions, emulsions, microcapsules, molecular complexes,microspheres, nanoparticles, patches, and liposomes.

As used herein, the phrases “parenteral administration” and“administered parenterally” as used herein mean modes of administrationother than enteral and topical administration, usually by injection, andincludes, without limitation, intravenous, intramuscular, intraarterial,intrathec al, intraventricular, intracapsular, intraorbital,intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous,subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal,intracerebrospinal, and intrasternal injection and infusion. The phrases“systemic administration,” “administered systemically”, “peripheraladministration” and “administered peripherally” as used herein mean theadministration therapeutic compositions other than directly into a tumorsuch that it enters the animal's system and, thus, is subject tometabolism and other like processes.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio. The phrase“pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in maintaining the activity of or carrying ortransporting the subject agents from one organ, or portion of the body,to another organ, or portion of the body. In addition to being“pharmaceutically acceptable” as that term is defined herein, eachcarrier must also be “acceptable” in the sense of being compatible withthe other ingredients of the formulation. The pharmaceutical formulationcomprising the one or more peptides as disclosed herein or a mutant,variant, analog or derivative thereof in combination with one or morepharmaceutically acceptable ingredients. The carrier can be in the formof a solid, semi-solid or liquid diluent, cream or a capsule. Thesepharmaceutical preparations are a further object of the invention.Usually the amount of active compounds is between 0.1-95% by weight ofthe preparation, preferably between 0.2-20% by weight in preparationsfor parenteral use and preferably between 1 and 50% by weight inpreparations for oral administration. For the clinical use of themethods of the present invention, targeted delivery composition of theinvention is formulated into pharmaceutical compositions orpharmaceutical formulations for parenteral administration, e.g.,intravenous; mucosal, e.g., intranasal; enteral, e.g., oral; topical,e.g., transdermal; ocular, e.g., via corneal scarification or other modeof administration. The pharmaceutical composition contains a compound ofthe invention in combination with one or more pharmaceuticallyacceptable ingredients. The carrier can be in the form of a solid,semi-solid or liquid diluent, cream or a capsule.

The term “pharmaceutically acceptable carriers” is intended to includeall solvents, diluents, or other liquid vehicle, dispersion orsuspension aids, surface active agents, isotonic agents, thickening oremulsifying agents, preservatives, solid binders, lubricants and thelike, as suited to the particular dosage form desired. Typically, suchcompounds are carried or transported from one organ, or portion of thebody, to another organ, or portion of the body. Each carrier must be“acceptable” in the sense of being compatible with the other ingredientsof the formulation and not injurious to the patient. Some examples ofmaterials which can serve as pharmaceutically acceptable carriersinclude: sugars, such as lactose, glucose and sucrose; starches, such ascorn starch and potato starch; cellulose, and its functionalderivatives, such as sodium carboxymethyl cellulose, ethyl cellulose andcellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients,such as cocoa butter and suppository waxes; oils, such as peanut oil,cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; glycols, such as propylene glycol; polyols, such asglycerin, sorbitol, mannitol and polyethylene glycol; esters, such asethyl oleate and ethyl laurate; agar; buffering agents, such asmagnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-freewater; isotonic saline; Ringer's solution; ethyl alcohol; phosphatebuffer solutions; and other non-toxic compatible substances employed inpharmaceutical formulations.

The term “pharmaceutical composition” is used herein refer tocompositions or formulations that usually comprise an excipient, such asa pharmaceutically acceptable carrier that is conventional in the artand that is suitable for administration to mammals, and preferablyhumans or human cells. Such compositions can be specifically formulatedfor administration via one or more of a number of routes, including butnot limited to, oral, ocular, parenteral, intravenous, intraarterial,subcutaneous, intranasal, sublingual, intraspinal,intracerebroventricular, and the like. In addition, compositions fortopical (e.g., oral mucosa, respiratory mucosa) and/or oraladministration can form solutions, suspensions, tablets, pills,capsules, sustained-release formulations, oral rinses, or powders, asknown in the art are described herein. The compositions also can includestabilizers and preservatives. For examples of carriers, stabilizers andadjuvants, University of the Sciences in Philadelphia (2005) Remington:The Science and Practice of Pharmacy with Facts and Comparisons, 21stEd.

In certain embodiments, the compounds of the present invention maycontain one or more acidic functional groups and, thus, are capable offorming pharmaceutically acceptable salts with pharmaceuticallyacceptable bases. The term “pharmaceutically acceptable salts, esters,amides, and prodrugs” as used herein refers to those carboxylate salts,amino acid addition salts, esters, amides, and prodrugs of the compoundsof the present invention which are, within the scope of sound medicaljudgment, suitable for use in contact with the tissues of patientswithout undue toxicity, irritation, allergic response, and the like,commensurate with a reasonable benefit/risk ratio, and effective fortheir intended use of the compounds of the invention. The term “salts”refers to the relatively non-toxic, inorganic and organic acid additionsalts of compounds of the present invention. These salts can be preparedin situ during the final isolation and purification of the compounds orby separately reacting the purified compound in its free base form witha suitable organic or inorganic acid and isolating the salt thus formed.These may include cations based on the alkali and alkaline earth metalssuch as sodium, lithium, potassium, calcium, magnesium and the like, aswell as nontoxic ammonium, quaternary ammonium, and amine cationsincluding, but not limited to ammonium, tetramethylanunonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine,ethylamine, and the like (see, e.g., Berge S. M., et al. (1977) J.Pharm. Sci. 66, 1, which is incorporated herein by reference).

The term “pharmaceutically acceptable esters” refers to the relativelynontoxic, esterified products of the compounds of the present invention.These esters can be prepared in situ during the final isolation andpurification of the compounds, or by separately reacting the purifiedcompound in its free acid form or hydroxyl with a suitable esterifyingagent. Carboxylic acids can be converted into esters via treatment withan alcohol in the presence of a catalyst. The term is further intendedto include lower hydrocarbon groups capable of being solvated underphysiological conditions, e.g., alkyl esters, methyl, ethyl and propylesters.

As used herein, “pharmaceutically acceptable salts or prodrugs” aresalts or prodrugs that are, within the scope of sound medical judgment,suitable for use in contact with the tissues of subject without unduetoxicity, irritation, allergic response, and the like, commensurate witha reasonable benefit/risk ratio, and effective for their intended use.

The term “prodrug” refers to compounds that are rapidly transformed invivo to yield the functionally active one or more peptides as disclosedherein or a mutant, variant, analog or derivative thereof. A thoroughdiscussion is provided in T. Higachi and V. Stella, “Pro-drugs as NovelDelivery Systems,” Vol. 14 of the A C. S. Symposium Series, and inBioreversible Carriers in: Drug Design, ed. Edward B. Roche, AmericanPharmaceutical Association and Pergamon Press, 1987, both of which arehereby incorporated by reference. As used herein, a prodrug is acompound that, upon in vivo administration, is metabolized or otherwiseconverted to the biologically, pharmaceutically or therapeuticallyactive form of the compound. A prodrug of the one or more peptides asdisclosed herein or a mutant, variant, analog or derivative thereof canbe designed to alter the metabolic stability or the transportcharacteristics of one or more peptides as disclosed herein or a mutant,variant, analog or derivative thereof, to mask side effects or toxicity,to improve the flavor of a compound or to alter other characteristics orproperties of a compound. By virtue of knowledge of pharmacodynamicprocesses and drug metabolism in vivo, once a pharmaceutically activeform of the one or more peptides as disclosed herein or a mutant,variant, analog or derivative thereof, those of skill in thepharmaceutical art generally can design prodrugs of the compound (see,e.g., Nogrady (1985) Medicinal Chemistry A Biochemical Approach, OxfordUniversity Press, N. Y., pages 388-392). Conventional procedures for theselection and preparation of suitable prodrugs are described, forexample, in “Design of Prodrugs,” ed. H. Bundgaard, Elsevier, 1985.Suitable examples of prodrugs include methyl, ethyl and glycerol estersof the corresponding acid.

Parenteral Compositions

The pharmaceutical composition may be administered parenterally byinjection, infusion, or implantation (subcutaneous, intravenous,intramuscular, intraperitoneal, or the like) in dosage forms,formulations, or via suitable delivery devices or implants containingconventional, non-toxic pharmaceutically acceptable carriers andadjuvants. The formulation and preparation of such compositions are wellknown to those skilled in the art of pharmaceutical formulation.

Compositions for parenteral use may be provided in unit dosage forms(e.g., in single-dose ampoules), or in vials containing several dosesand in which a suitable preservative may be added (see below). Thecomposition may be in form of a solution, a suspension, an emulsion, aninfusion device, or a delivery device for implantation, or it may bepresented as a dry powder to be reconstituted with water or anothersuitable vehicle before use. Apart from the active agent(s), thecomposition may include suitable parenterally acceptable carriers and/orexcipients. The active agent(s) may be incorporated into microspheres,microcapsules, nanoparticles, liposomes, or the like for controlledrelease. Furthermore, the composition may include suspending,solubilizing, stabilizing, pH-adjusting agents, tonicity adjustingagents, and/or dispersing agents.

As indicated above, the pharmaceutical compositions according to theinvention may be in a form suitable for sterile injection. To preparesuch a composition, the suitable active agent(s) are dissolved orsuspended in a parenterally acceptable liquid vehicle. Among acceptablevehicles and solvents that may be employed are water, water adjusted toa suitable pH by addition of an appropriate amount of hydrochloric acid,sodium hydroxide or a suitable buffer, 1,3-butanediol, Ringer'ssolution, dextrose solution, and isotonic sodium chloride solution. Theaqueous formulation may also contain one or more preservatives (e.g.,methyl, ethyl or n-propyl p-hydroxybenzoate). In cases where one of thecompounds is only sparingly or slightly soluble in water, a dissolutionenhancing or solubilizing agent can be added, or the solvent may include10-60% w/w of propylene glycol or the like.

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfate, sodium sulfite and the like;oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Formulations of the present invention include those suitable forintravenous, oral, nasal, topical, transdermal, buccal, sublingual,rectal, vaginal and/or parenteral administration. The formulations mayconveniently be presented in unit dosage form and may be prepared by anymethods well known in the art of pharmacy. The amount of activeingredient which can be combined with a carrier material to produce asingle dosage form will generally be that amount of the compound whichproduces a therapeutic effect.

Methods of preparing these formulations or compositions include the stepof bringing into association a compound of the present invention withthe carrier and, optionally, one or more accessory ingredients. Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association a compound of the present invention withliquid carriers, or finely divided solid carriers, or both, and then, ifnecessary, shaping the product.

Formulations of the invention suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of a compound of thepresent invention as an active ingredient. A compound of the presentinvention may also be administered as a bolus, electuary or paste.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more peptides as disclosed herein or amutant, variant, analog or derivative thereof in combination with one ormore pharmaceutically acceptable sterile isotonic aqueous or nonaqueoussolutions, dispersions, suspensions or emulsions, or sterile powderswhich may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain antioxidants, buffers,bacteriostats, solutes which render the formulation isotonic with theblood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions comprising one or morepeptides as disclosed herein or a mutant, variant, analog or derivativethereof include water, ethanol, polyols (such as glycerol, propyleneglycol, polyethylene glycol, and the like), and suitable mixturesthereof, vegetable oils, such as olive oil, and injectable organicesters, such as ethyl oleate. Proper fluidity can be maintained, forexample, by the use of coating materials, such as lecithin, by themaintenance of the required particle size in the case of dispersions,and by the use of surfactants.

These compositions can also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption such as aluminum monostearate andgelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsulated matrices ofthe subject compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug, such as one ormore peptides as disclosed herein or a mutant, variant, analog orderivative thereof in liposomes or microemulsions which are compatiblewith body tissue.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically acceptable dosage forms by conventional methodsknown to those of ordinary skill in the art.

Controlled Release Parenteral Compositions

Controlled release parenteral compositions may be in form of aqueoussuspensions, microspheres, microcapsules, magnetic microspheres, oilsolutions, oil suspensions, or emulsions. The composition may also beincorporated in biocompatible carriers, liposomes, nanoparticles,implants, or infusion devices.

Materials for use in the preparation of microspheres and/ormicrocapsules are, e.g., biodegradable/bioerodible polymers such aspolygalactia poly-(isobutyl cyanoacrylate),poly(2-hydroxyethyl-L-glutamnine), poly(lactic acid), polyglycolic acid,and mixtures thereof. Biocompatible carriers that may be used whenformulating a controlled release parenteral formulation arecarbohydrates (e.g., dextrans), proteins (e.g., albumin), lipoproteins,or antibodies. Materials for use in implants can be nonbiodegradable(e.g., polydimethyl siloxane) or biodegradable (e.g.,poly(caprolactone), poly(lactic acid), poly(glycolic acid) or poly(orthoesters)) or combinations thereof.

Solid Dosage Forms for Oral Use

Formulations for oral use include tablets containing the activeingredient(s) in a mixture with non-toxic pharmaceutically acceptableexcipients, and such formulations are known to the skilled artisan(e.g., U.S. Pat. Nos.: 5,817,307; 5,824,300; 5,830,456; 5,846,526;5,882,640; 5,910,304; 6,036,949; 6,036,949; and 6,372,218 herebyincorporated by reference). These excipients may be, for example, inertdiluents or fillers (e.g., sucrose, sorbitol, sugar, mannitol,microcrystalline cellulose, starches including potato starch, calciumcarbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate,or sodium phosphate); granulating and disintegrating agents (e.g.,cellulose derivatives including microcrystalline cellulose, starchesincluding potato starch, croscarmellose sodium, alginates, or alginicacid); binding agents (e.g., sucrose, glucose, sorbitol, acacia, alginicacid, sodium alginate, gelatin, starch, pregelatinized starch,microcrystalline cellulose, magnesium aluminum silicate,carboxymethylcellulose sodium, methylcellulose, hydroxypropylmethylcellulose, ethylcellulose, polyvinylpyrrolidone, or polyethyleneglycol); and lubricating agents, glidants, and anti-adhesives (e.g.,magnesium stearate, zinc stearate, stearic acid, silicas, hydrogenatedvegetable oils, or talc). Other pharmaceutically acceptable excipientscan be colorants, flavoring agents, plasticizers, humectants, bufferingagents, and the like.

The tablets may be uncoated or they may be coated by known techniques,optionally to delay disintegration and absorption in thegastrointestinal tract and thereby providing a sustained action over alonger period. The coating may be adapted to release the protein in apredetermined pattern (e.g., in order to achieve a controlled releaseformulation) or it may be adapted not to release the agent(s) untilafter passage of the stomach (enteric coating). The coating may be asugar coating, a film coating (e.g., based on hydroxypropylmethylcellulose, methylcellulose, methyl hydroxyethylcellulose,hydroxypropylcellulose, carboxymethylcellulose, acrylate copolymers,polyethylene glycols and/or polyvinylpyrrolidone), or an enteric coating(e.g., based on methacrylic acid copolymer, cellulose acetate phthalate,hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcelluloseacetate succinate, polyvinyl acetate phthalate, shellac, and/orethylcellulose). Furthermore, a time delay material such as, e.g.,glyceryl monostearate or glyceryl distearate, may be employed.

The solid tablet compositions may include a coating adapted to protectthe composition from unwanted chemical changes, (e.g., chemicaldegradation prior to the release of the active substances). The coatingmay be applied on the solid dosage form in a similar manner as thatdescribed in Encyclopedia of Pharmaceutical Technology, supra.

The compositions of the invention may be mixed together in the tablet,or may be partitioned. In one example, a first agent is contained on theinside of the tablet, and a second agent is on the outside, such that asubstantial portion of the second agent is released prior to the releaseof the first agent.

Formulations for oral use may also be presented as chewable tablets, oras hard gelatin capsules wherein the active ingredient is mixed with aninert solid diluent (e.g., potato starch, lactose, microcrystallinecellulose, calcium carbonate, calcium phosphate, or kaolin), or as softgelatin capsules wherein the active ingredient is mixed with water or anoil medium, for example, peanut oil, liquid paraffin, or olive oil.Powders and granulates may be prepared using the ingredients mentionedabove under tablets and capsules in a conventional manner using, e.g., amixer, a fluid bed apparatus, or spray drying equipment.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules and the like), theactive ingredient is mixed with one or more pharmaceutically acceptablecarriers, such as sodium citrate or dicalcium phosphate, and/or any ofthe following: fillers or extenders, such as starches, lactose, sucrose,glucose, mannitol, and/or silicic acid; binders, such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,sucrose and/or acacia; humectants, such as glycerol; disintegratingagents, such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate; solutionretarding agents, such as paraffin; absorption accelerators, such asquatemary ammonium compounds; wetting agents, such as, for example,cetyl alcohol and glycerol monostearate; absorbents, such as kaolin andbentonite clay; lubricants, such a talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate, andmixtures thereof; and coloring agents. In the case of capsules, tabletsand pills, the pharmaceutical compositions may also comprise bufferingagents. Solid compositions of a similar type may also be employed asfillers in soft and hard-filled gelatin capsules using such excipientsas lactose or milk sugars, as well as high molecular weight polyethyleneglycols and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved in sterile water, or some other sterile injectable mediumimmediately before use. These compositions may also optionally containopacifying agents and may be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain portion ofthe gastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active ingredient can also be in micro-encapsulated form,if appropriate, with one or more of the above-described excipients. Inone aspect, a solution of resolvin and/or protectin or precursor oranalog thereof can be administered as eye drops for ocularneovascularization or ear drops to treat otitis.

Oral administration of the peptides useful according to this inventionhave been shown to work as discussed, e.g., in U.S. Pat. No. 8,975,224.

Oral administration of peptides has been shown to work for other proteinor peptide drugs as well. For example, oral administration of ananti-CD3 antibody has been shown to work in treatment of, for example,diabetes (Ishikawa et al. Diabetes. 2007 August; 56(8):2103-9. Epub Apr.24, 2007), and autoimmune encephalomyelitis (Ochi et al. Nat Med. 2006June; 12(6):627-35. Epub May 21, 2006). Without wishing to be bound by atheory, we suggest that this is possible via the gut associated lymphoidtissue (GALT). Accordingly, in some aspects of all the embodiments ofthe invention, the formulation of the peptides is oral formulation, andthe methods are performed by administering the peptides orally.

Liquid dosage forms for oral administration of the compounds of theinvention include pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs.

In addition to the active ingredient, the liquid dosage forms maycontain inert diluents commonly used in the art, such as, for example,water or other solvents, solubilizing agents and emulsifiers, such asethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils(in particular, cottonseed, groundnut, corn, germ, olive, castor andsesame oils), glycerol, tetrahydrofulyl alcohol, polyethylene glycolsand fatty acid esters of sorbitan, and mixtures thereof. Besides inertdiluents, the oral compositions can also include adjuvants such aswetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Dosage forms for the topical or transdermal administration of one ormore peptides as disclosed herein or a mutant, variant, analog orderivative thereof include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches and inhalants. The active compound maybe mixed under sterile conditions with a pharmaceutically acceptablecarrier, and with any preservatives, buffers, or propellants, which maybe required.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of this invention, excipients, such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof. Powders and sprays cancontain, in addition to a compound of this invention, excipients such aslactose, talc, silicic acid, aluminum hydroxide, calcium silicates andpolyamide powder, or mixtures of these substances. Sprays canadditionally contain customary propellants, such aschlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, suchas butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of the compounds (resolvins and/or protectins and/or precursorsor analogues thereof) of the present invention to the body. Such dosageforms can be made by dissolving or dispersing the compound in the propermedium. Absorption enhancers can also be used to increase the flux ofthe compound across the skin. The rate of such flux can be controlled byeither providing a rate controlling membrane or dispersing the activecompound in a polymer matrix or gel. In another aspect, biodegradable orabsorbable polymers can provide extended, often localized, release ofpolypeptide agents. The potential benefits of an increased half-life orextended release for a therapeutic agent are clear. A potential benefitof localized release is the ability to achieve much higher localizeddosages or concentrations, for greater lengths of time, relative tobroader systemic administration, with the potential to also avoidpossible undesirable side effects that may occur with systemicadministration.

Bioabsorbable polymeric matrix suitable for delivery of the one or morepeptides as disclosed herein or a mutant, variant, analog or derivativethereof can be selected from a variety of synthetic bioabsorbablepolymers, which are described extensively in the literature. Suchsynthetic bioabsorbable, biocompatible polymers, which may releaseproteins over several weeks or months can include, for example,poly-α-hydroxy acids (e.g. polylactides, polyglycolides and theircopolymers), polyanhydrides, polyorthoesters, segmented block copolymersof polyethylene glycol and polybutylene terephtalate (Polyactive™),tyrosine derivative polymers or poly(ester-amides). Suitablebioabsorbable polymers to be used in manufacturing of drug deliverymaterials and implants are discussed e.g. in U.S. Pat. Nos. 4,968,317,5,618,563, among others, and in “Biomedical Polymers” edited by S. W.Shalaby, Carl Hanser Verlag, Munich, Vienna, N.Y., 1994 and in manyreferences cited in the above publications. The particular bioabsorbablepolymer that should be selected will depend upon the particular patientthat is being treated.

Gene Therapy

One or more peptides as disclosed herein or a mutant, variant, analog orderivative thereof can be effectively used in treatment by gene therapy.See, generally, for example, U.S. Pat. No. 5,399,346, which isincorporated herein by reference. The general principle is to introducethe polynucleotide into a target cell in a patient.

Entry into the cell is facilitated by suitable techniques known in theart such as providing the polynucleotide in the form of a suitablevector, or encapsulation of the polynucleotide in a liposome.

A desired mode of gene therapy is to provide the polynucleotide in sucha way that it will replicate inside the cell, enhancing and prolongingthe desired effect. Thus, the polynucleotide is operably linked to asuitable promoter, such as the natural promoter of the correspondinggene, a heterologous promoter that is intrinsically active in liver,neuronal, bone, muscle, skin, joint, or cartilage cells, or aheterologous promoter that can be induced by a suitable agent.

Expression vectors compatible with eukaryotic cells, preferably thosecompatible with vertebrate cells, can be used to produce recombinantconstructs for the expression of one or more peptides as disclosedherein or a mutant, variant, analog or derivative thereof, includingfusion proteins with one or more peptides as disclosed herein or amutant, variant, analog or derivative thereof. Eukaryotic cellexpression vectors are well known in the art and are available fromseveral commercial sources. Typically, such vectors are providedcontaining convenient restriction sites for insertion of the desired DNAsegment. These vectors can be viral vectors such as adenovirus,adeno-associated virus, pox virus such as an orthopox (vaccinia andattenuated vaccinia), avipox, lentivirus, murine moloney leukemia virus,etc. Alternatively, plasmid expression vectors can be used.

Viral vector systems which can be utilized in the present inventioninclude, but are not limited to, (a) adenovirus vectors; (b) retrovirusvectors; (c) adeno-associated virus vectors; (d) herpes simplex virusvectors; (e) SV 40 vectors; (f) polyoma virus vectors; (g) papillomavirus vectors; (h) picornavirus vectors; (i) pox virus vectors such asan orthopox, e.g., vaccinia virus vectors or avipox, e.g. canary pox orfowl pox; and (j) a helper-dependent or gutless adenovirus. In apreferred embodiment, the vector is an adenovirus. Replication-defectiveviruses can also be advantageous.

The vector may or may not be incorporated into the cells genome. Theconstructs may include viral sequences for transfection, if desired.Alternatively, the construct may be incorporated into vectors capable ofepisomal replication, e.g. EPV and EBV vectors.

By “operably linked” is meant that a nucleic acid molecule and one ormore regulatory sequences (e.g., a promoter) are connected in such awayas to permit expression and/or secretion of the product (e.g., aprotein) of the nucleic acid molecule when the appropriate molecules(e.g., transcriptional activator proteins) are bound to the regulatorysequences. Stated another way, the term “operatively linked” as usedherein refers to the functional relationship of the nucleic acidsequences with regulatory sequences of nucleotides, such as promoters,enhancers, transcriptional and translational stop sites, and othersignal sequences. For example, operative linkage of nucleic acidsequences, typically DNA, to a regulatory sequence or promoter regionrefers to the physical and functional relationship between the DNA andthe regulatory sequence or promoter such that the transcription of suchDNA is initiated from the regulatory sequence or promoter, by an RNApolymerase that specifically recognizes, binds and transcribes the DNAIn order to optimize expression and/or in vitro transcription, it may benecessary to modify the regulatory sequence for the expression of thenucleic acid or DNA in the cell type for which it is expressed. Thedesirability of, or need of, such modification may be empiricallydetermined. An operatively linked polynucleotide which is to beexpressed typically includes an appropriate start signal (e.g., ATG) andmaintains the correct reading frame to permit expression of thepolynucleotide sequence under the control of the expression controlsequence, and production of the desired polypeptide encoded by thepolynucleotide sequence.

As used herein, the terms “promoter” or “promoter region” or “promoterelement” have been defined herein, refers to a segment of a nucleic acidsequence, typically but not limited to DNA or RNA or analogues thereof,that controls the transcription of the nucleic acid sequence to which itis operatively linked. The promoter region includes specific sequencesthat are sufficient for RNA polymerase recognition, binding andtranscription initiation. This portion of the promoter region isreferred to as the promoter. In addition, the promoter region includessequences which modulate this recognition, binding and transcriptioninitiation activity of RNA polymerase. These sequences may be ds-actingor may be responsive to trans-acting factors. Promoters, depending uponthe nature of the regulation may be constitutive or regulated.

The term “regulatory sequences” is used interchangeably with “regulatoryelements” herein refers element to a segment of nucleic acid, typicallybut not limited to DNA or RNA or analogues thereof, that modulates thetranscription of the nucleic acid sequence to which it is operativelylinked, and thus act as transcriptional modulators. Regulatory sequencesmodulate the expression of gene and/or nucleic acid sequence to whichthey are operatively linked. Regulatory sequence often comprise“regulatory elements” which are nucleic acid sequences that aretranscription binding domains and are recognized by the nucleicacid-binding domains of transcriptional proteins and/or transcriptionfactors, repressors or enhancers etc. Typical regulatory sequencesinclude, but are not limited to, transcriptional promoters, induciblepromoters and transcriptional elements, an optional operate sequence tocontrol transcription, a sequence encoding suitable mRNA ribosomalbinding sites, and sequences to control the termination of transcriptionand/or translation. Included in the term “regulatory elements” arenucleic acid sequences such as initiation signals, enhancers, andpromoters, which induce or control transcription of protein codingsequences with which they are operatively linked. In some examples,transcription of a recombinant gene is under the control of a promotersequence (or other transcriptional regulatory sequence) which controlsthe expression of the recombinant gene in a cell-type in whichexpression is intended. It will also be understood that the recombinantgene can be under the control of transcriptional regulatory sequenceswhich are the same or which are different from those sequences whichcontrol transcription of the naturally-occurring form of a protein. Insome instances the promoter sequence is recognized by the syntheticmachinery of the cell, or introduced synthetic machinery, required forinitiating transcription of a specific gene.

Regulatory sequences can be a single regulatory sequence or multipleregulatory sequences, or modified regulatory sequences or fragmentsthereof. Modified regulatory sequences are regulatory sequences wherethe nucleic acid sequence has been changed or modified by some means,for example, but not limited to, mutation, methylation etc.

Regulatory sequences useful in the methods as disclosed herein arepromoter elements which are sufficient to render promoter-dependent geneexpression controllable for cell type-specific, tissue-specific orinducible by external signals or agents (e.g. enhancers or repressors);such elements may be located in the 5′ or 3′ regions of the native gene,or within an intron.

As used herein, the term “tissue-specific promoter” means a nucleic acidsequence that serves as a promoter, i.e., regulates expression of aselected nucleic acid sequence operably linked to the promoter, andwhich selectively affects expression of the selected nucleic acidsequence in specific cells of a tissue.

In some embodiments, it can be advantageous to direct expression of oneor more peptides as disclosed herein or a mutant, variant, analog orderivative thereof in a tissue- or cell-specific manner. Muscle-specificexpression can be achieved, for example, using the skeletal muscle MKCpromoter (as disclosed in U.S. Patent Application WO2007/100722, whichis incorporated herein by reference), or other muscle-specificpromoters, such as α-myosin heavy chain, myosin light chain-2 (which isspecific for skeletal muscle (Shani et al., Nature, 314;283-86, 1985),gonadotrophic releasing hormone gene control region which is active inthe hypothalamus (Mason et al, Science, 234;1372-78, 1986), and smoothmuscle promoter SM22a, which are all commonly known in the art.

The term “constitutively active promoter” refers to a promoter of a genewhich is expressed at all times within a given cell. Exemplary promotersfor use in mammalian cells include cytomegalovirus (CMV), and for use inprokaryotic cells include the bacteriophage T7 and T3 promoters, and thelike. The term “inducible promoter” refers to a promoter of a gene whichcan be expressed in response to a given signal, for example addition orreduction of an agent. Non-limiting examples of an inducible promoterare “tet-on” and “tet-off” promoters, or promoters that are regulated ina specific tissue type.

In a specific embodiment, viral vectors that contain nucleic acidsequences encoding the one or more peptides as disclosed herein or amutant, variant, analog or derivative thereof are used. For example, aretroviral vector can be used (see Miller et al., Meth. Enzymol.217:581-599 (1993)). These retroviral vectors contain the componentsnecessary for the correct packaging of the viral genome and integrationinto the host cell DNA More detail about retroviral vectors can be foundin Boesen et al., Biotherapy 6:291-302 (1994), which describes the useof a retroviral vector to deliver the mdrl gene to hematopoietic stemcells in order to make the stem cells more resistant to chemotherapy.Other references illustrating the use of retroviral vectors in genetherapy are: Clowes et al., J. Clin. Invest. 93:644-651 (1994); Kiem etal., Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics andDevel. 3:110-114 (1993).

The production of a recombinant retroviral vector carrying a gene ofinterest is typically achieved in two stages. First, sequence encodingone or more peptides as disclosed herein or a mutant, variant, analog orderivative thereof can be inserted into a retroviral vector whichcontains the sequences necessary for the efficient expression of themetabolic regulators (including promoter and/or enhancer elements whichcan be provided by the viral long terminal repeats (LTRs) or by aninternal promoter/enhancer and relevant splicing signals), sequencesrequired for the efficient packaging of the viral RNA into infectiousvirions (e.g., a packaging signal (Psi), a tRNA primer binding site(-PBS), a 3′ regulatory sequence required for reverse transcription(+PBS)), and a viral LTRs). The LTRs contain sequences required for theassociation of viral genomic RNA, reverse transcriptase and integrasefunctions, and sequences involved in directing the expression of thegenomic RNA to be packaged in viral particles.

Following the construction of the recombinant retroviral vector, thevector DNA is introduced into a packaging cell line. Packaging celllines provide viral proteins required in trans for the packaging ofviral genomic RNA into viral particles having the desired host range(e.g., the viral-encoded core (gag), polymerase (pol) and envelope (env)proteins). The host range is controlled, in part, by the type ofenvelope gene product expressed on the surface of the viral particle.Packaging cell lines can express ecotrophic, amphotropic or xenotropicenvelope gene products. Alternatively, the packaging cell line can lacksequences encoding a viral envelope (env) protein. In this case, thepackaging cell line can package the viral genome into particles whichlack a membrane-associated protein (e.g., an env protein). To produceviral particles containing a membrane-associated protein which permitsentry of the virus into a cell, the packaging cell line containing theretroviral sequences can be transfected with sequences encoding amembrane-associated protein (e.g., the G protein of vesicular stomatitisvirus (VSV)). The transfected packaging cell can then produce viralparticles which contain the membrane-associated protein expressed by thetransfected packaging cell line; these viral particles which containviral genomic RNA derived from one virus encapsidated by the envelopeproteins of another virus are said to be pseudotyped virus particles.

Adenoviruses are other viral vectors that can be used in gene therapy.Adenoviruses are especially attractive vehicles for delivering genes torespiratory epithelia. Adenoviruses naturally infect respiratoryepithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson, CurrentOpinion in Genetics and Development 3:499-503 (1993) present a review ofadenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10(1994) demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Another preferred viralvector is a pox virus such as a vaccinia virus, for example anattenuated vaccinia such as Modified Virus Ankara (MVA) or NYVAC, anavipox such as fowl pox or canary pox. Other instances of the use ofadenoviruses in gene therapy can be found in Rosenfeld et al., Science252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992);Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT PublicationW094/12649; and Wang, et al., Gene Therapy 2:775-783 (1995). In anotherembodiment, lentiviral vectors are used, such as the HIV based vectorsdescribed in U.S. Pat. Nos. 6,143,520; 5,665,557; and 5,981,276, whichare herein incorporated by reference. Use of Adeno-associated virus(AAV) vectors is also contemplated (Walsh et al., Proc. Soc. Exp. Biol.Med. 204:289-300 (1993); and U.S. Pat. No. 5,436,146, which areincorporated herein by reference).

Another approach to gene therapy involves transferring a gene to cellsin tissue culture by such methods as electroporation, lipofection,calcium phosphate mediated transfection, or viral infection. Usually,the method of transfer includes the transfer of a selectable marker tothe cells. The cells are then placed under selection to isolate thosecells that have taken up and are expressing the transferred gene. Thosecells are then delivered to a patient.

U.S. Pat. No. 5,676,954 (which is herein incorporated by reference)reports on the injection of genetic material, complexed with cationicliposome carriers, into mice. U.S. Pat. Nos. 4,897,355, 4,946,787,5,049,386, 5,459,127, 5,589,466, 5,693,622, 5,580,859, 5,703,055, andinternational publication NO: WO 94/9469 (which are herein incorporatedby reference) provide cationic lipids for use in transfecting DNA intocells and mammals. U.S. Pat. Nos. 5,589,466, 5,693,622, 5,580,859,5,703,055, and international publication NO: WO 94/9469 (which areherein incorporated by reference) provides methods for deliveringDNA-cationic lipid complexes to mammals. Such cationic lipid complexesor nanoparticles can also be used to deliver protein.

A gene or nucleic acid sequence can be introduced into a target cell byany suitable method. For example, one or more peptides as disclosedherein or a mutant, variant, analog or derivative thereof constructs canbe introduced into a cell by transfection (e.g., calcium phosphate orDEAE-dextran mediated transfection), lipofection, electroporation,microinjection (e.g., by direct injection of naked DNA), biolistics,infection with a viral vector containing a muscle related transgene,cell fusion, chromosome-mediated gene transfer, microcell-mediated genetransfer, nuclear transfer, and the like. A nucleic acid encoding one ormore peptides as disclosed herein or a mutant, variant, analog orderivative thereof can be introduced into cells by electroporation (see,e.g., Wong and Neumann, Biochem. Biophys. Res. Commun. 107:584-87(1982)) and biolistics (e.g., a gene gun; Johnston and Tang, MethodsCell Biol. 43 Pt A353-65 (1994); Fynan et al., Proc. Natl. Acad. Sci.USA 90:11478-82 (1993)).

In certain embodiments, a gene or nucleic acid sequence encoding one ormore peptides as disclosed herein or a mutant, variant, analog orderivative thereof can be introduced into target cells by transfectionor lipofection. Suitable agents for transfection or lipofection include,for example, calcium phosphate, DEAE dextran, lipofectin, lipfectamine,DIMRIE C, Superfect, and Effectin (Qiagen), unifectin, maxifectin,DOTMA, DOGS (Transfectam; dioctadecylamidoglycylspermine), DOPE(1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), DOTAP(1,2-dioleoyl-3-trimethylammonium propane), DDAB (dimethyldioctadecylammonium bromide), DHDEAB(N,N-di-n-hexadecyl-N,N-dihydroxyethyl ammonium bromide), HDEAB(N-n-hexadecyl-N,N-dihydroxyethylammonium bromide), polybrene,poly(ethylenimine) (PEI), and the like. (See, e.g., Banerjee et al.,Med. Chem. 42:4292-99 (1999); Godbey et al., Gene Ther. 6:1380-88(1999); Kichler et al., Gene Ther. 5:855-60 (1998); Birchaa et al., J.Pharm. 183:195-207 (1999), incorporated herein by reference in theirentireties).

Methods known in the art for the therapeutic delivery of agents such asproteins and/or nucleic acids can be used for the delivery of apolypeptide or nucleic acid encoding one or more peptides as disclosedherein or a mutant, variant, analog or derivative thereof, e.g.,cellular transfection, gene therapy, direct administration with adelivery vehicle or pharmaceutically acceptable carrier, indirectdelivery by providing recombinant cells comprising a nucleic acidencoding a targeting fusion polypeptide of the invention.

Various delivery systems are known and can be used to directlyadminister therapeutic polypeptides such as the one or more peptides asdisclosed herein or a mutant, variant, analog or derivative thereofand/or a nucleic acid encoding one or more peptides as disclosed hereinor a mutant, variant, analog or derivative thereof, e.g., encapsulationin liposomes, microparticles, microcapsules, recombinant cells capableof expressing the compound, and receptor-mediated endocytosis (see,e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432). Methods ofintroduction can be enteral or parenteral and include but are notlimited to intradermal, intramuscular, intraperitoneal, intravenous,subcutaneous, pulmonary, intranasal, intraocular, epidural, and oralroutes. The agents may be administered by any convenient route, forexample by infusion or bolus injection, by absorption through epithelialor mucocutaneous linings (e.g., oral mucosa, rectal and intestinalmucosa, etc.) and may be administered together with other biologicallyactive agents. Administration can be systemic or local.

In a specific embodiment, it may be desirable to administer thepharmaceutical compositions of the invention locally to the area in needof treatment; this may be achieved, for example, and not by way oflimitation, by local infusion during surgery, topical application, e.g.,by injection, by means of a catheter, or by means of an implant, theimplant being of a porous, non-porous, or gelatinous material, includingmembranes, such as sialastic membranes, fibers, or commercial skinsubstitutes.

In another embodiment, the active agent can be delivered in a vesicle,in particular a liposome (see Langer (1990) Science 249:1527-1533). Inyet another embodiment, the active agent can be delivered in acontrolled release system. In one embodiment, a pump may be used (seeLanger (1990) supra). In another embodiment, polymeric materials can beused (see Howard et al. (1989) J. Neurosurg. 71:105).

Thus, a wide variety of gene transfer/gene therapy vectors andconstructs are known in the art. These vectors are readily adapted foruse in the methods of the present invention. By the appropriatemanipulation using recombinant DNA/molecular biology techniques toinsert an operatively linked polypeptide encoding nucleic acid segmentinto the selected expression/delivery vector, many equivalent vectorsfor the practice of the methods described herein can be generated.

Other Embodiments

From the foregoing description, it will be apparent that variations andmodifications may be made to the invention described herein to adopt itto various usages and conditions. Such embodiments are also within thescope of the following claims.

The disclosure also contemplates an article of manufacture, which is alabeled container for providing the one or more peptides as disclosedherein or a mutant, variant, analog or derivative thereof. An article ofmanufacture comprises packaging material and a pharmaceutical agent ofthe one or more peptides as disclosed herein or a mutant, variant,analog or derivative thereof, contained within the packaging material.

The pharmaceutical agent in an article of manufacture is any of thecompositions of the present invention suitable for providing the one ormore peptides as disclosed herein or a mutant, variant, analog orderivative thereof and formulated into a pharmaceutically acceptableform as described herein according to the disclosed indications. Thus,the composition can comprise the one or more peptides as disclosedherein or a mutant, variant, analog or derivative thereof or a DNAmolecule which is capable of expressing such a peptide.

The article of manufacture contains an amount of pharmaceutical agentsufficient for use in treating a condition indicated herein, either inunit or multiple dosages. The packaging material comprises a label whichindicates the use of the pharmaceutical agent contained therein.

The label can further include instructions for use and relatedinformation as may be required for marketing. The packaging material caninclude container(s) for storage of the pharmaceutical agent.

As used herein, the term packaging material refers to a material such asglass, plastic, paper, foil, and the like capable of holding withinfixed means a pharmaceutical agent. Thus, for example, the packagingmaterial can be plastic or glass vials, laminated envelopes and the likecontainers used to contain a pharmaceutical composition including thepharmaceutical agent.

In preferred embodiments, the packaging material includes a label thatis a tangible expression describing the contents of the article ofmanufacture and the use of the pharmaceutical agent contained therein.

EXAMPLES Example 1 The Use of SP163M Peptide for Treatment of AcuteMyocardial Infarction

SP16 peptide is a short fragment derived from the circulating serumprotein, alpha-1-antitrypsin (AAT). The US FDA has approved the use ofthree alpha 1-antitrypsin products derived from a human plasma:Prolastin, Zemaira, and Aralast.

As described herein, SP163M peptide contains the reportedanti-inflammatory properties of the parental AAT. AAT has shownpreliminary safety and efficacy in blocking the inflammatory reactionthat occurs after AMI in a pilot clinical study conducted at VirginiaCommonwealth University (VCU). Furthermore, SP163M has shown equal orbetter anti-inflammatory activity in preclinical models of AMI. We arecollaborating with researchers at VCU to rapidly develop SP16 fortreatment of AMI which represent a major unmet medical need in the USand worldwide.

Acute myocardial infarction (AMI) remains a major cause of morbidity andmortality in the US and worldwide. Despite current strategies for earlyreperfusion, many patients die early during the course, and those whosurvive are at risk for dying later from adverse cardiac remodeling,heart failure, and sudden death. Numerous publications emphasize thecorrelation between the size of the infarct in AMI and the probabilityto progress to heart failure in the next 2-5 years post AMI.

Patients presenting with ST-segment elevation (STEMI) are atparticularly high risk for adverse cardiac remodeling, heart failure,and in-hospital and long-term mortality. Although there have beenconsiderable improvements in the treatment of STEMI, the reduction inearly mortality has been associated with an increasing incidence ofheart failure after STEMI.¹ This likely reflects more high risk patientssurviving the index event as well as the aging of the population and theepidemics of hypertension and diabetes. Within 30 days of STEMI, morethan 20% of survivors are diagnosed with heart failure, a diseaseassociated with high morbidity, disability, and mortality.

Heart failure is indeed a major public health problem affectingapproximately 5 million Americans with 500,000 new cases per year. Incontrast to other cardiovascular disease, the incidence and prevalenceof heart failure continue to increase and heart failure is now theleading cause of hospitalization for people aged 65 years, a segment ofthe population that is also rapidly growing. Although survival after theonset of heart failure is also improved, current therapies may slow butnot halt the progression of the disease. With the limitations tofunctional capacity, the progressive symptoms of dyspnea and fatigue,the frequent hospital admissions and the economic consequences of lostproductivity and increasing costs of medical care, heart failure imposesa significant burden on healthcare.

There is an urgent need to develop additional treatments to minimize theinfarct size and prevent heart failure after AMI.² The current treatmentin STEMI includes prompt reperfusion of the ischemic myocardium byrestoration of the coronary artery patency (i.e. angioplasty orfibrinolysis), prevention of re-occlusion (i.e. antiplatelet andanticoagulants), and neuro-hormonal blockade (i.e.renin-angiotensin-aldosterone and adrenergic blockers). While each ofthese interventions provide incremental benefit and significantly reducemorbidity and mortality, the incidence of heart failure after STEMI hascontinued to rise, implying that the current treatment paradigm stillmisses one or more key pathophysiologic mechanisms.

Determining the mechanisms by which unfavorable cardiac remodeling andheart failure progress despite optimal treatment is thus a critical stepin the search for novel interventions, with the ultimate goal ofreducing the incidence, burden, and mortality of heart failure afterSTEMI.

A close interplay exists between inflammation, adverse cardiacremodeling and heart failure after AMI. Acute myocardial ischemia andinfarction initiate an intense inflammatory response within themyocardium.³ Leukocytes infiltrate the damaged myocardium to coordinatetissue repair and infarct healing, leading to newly formed vessels andreparative fibrosis. Thus, inflammation is necessary for infarcthealing, but uncontrolled inflammation is responsible for further damageto the heart and non-functional healing which are the basis for adversecardiac remodeling and heart failure. In experimental animal models, thedegree of the inflammatory response determines adverse cardiacremodeling, independent of infarct size.³ In patients with AMI, theintensity of the inflammatory response, reflected in levels ofcirculating biomarkers, predicts adverse cardiac remodeling, heartfailure and death.⁴ C-reactive protein (an acute phase reactant) areincreased in AMI and predict outcome.

Modulation of the inflammatory response therefore represents a targetfor intervention. While previous attempts to modulate the inflammatoryresponse have failed, we propose a novel and substantially differentapproach to modulating the inflammatory response using SP163M peptide a17-mer modified derivative of Alpha-1 Anti-Trypsin (AAT).⁵ AAT is anaturally occurring anti-inflammatory protein abundant in the plasma,exerting powerful cyto-protective effects in endothelial cells,cardiomyocytes and fibroblasts.⁵

In an experimental model of acute myocardial infarction (AMI), AATadministered at the onset of ischemia or at time of reperfusion led to asignificantly smaller infarct size and more favorable cardiac healingand remodeling (FIG. 3A-3D).⁶

While there may be more than one mechanism by which AAT improves cardiacfunction during AMI, an inhibition of the production ofInterleukin-1IL-1) is proposed.⁶⁻⁷ This is relevant because IL-1inhibition has been shown to be safe and efficacious in limitingmyocardial damage during AMI both in experimental studies and in 2 smallpilot clinical trials that were completed at VCU.⁸⁻¹⁰

In order to examine the effects of SP163M in AMI, the peptide was testedin the same mouse model previously used for AAT. Briefly, adult male ICRmice underwent coronary artery ligation inducing myocardial ischemia for30 minutes followed by reperfusion. SP16 at 100 μg dose or matchingvolume of NaCl 0.9% solution was given at time of reperfusion. SP16significantly reduces by >50% the myocardial infarct size (FIG. 4A),measured as percentage of infarcted left ventricle (appearing white atthe triphenyltetrazolium chloride stain) on the whole left ventricle,with viable myocardium appearing bright red. The plasma levels ofcardiac specific troponin I, a biomarker of myocardial necrosis, wasalso significantly reduced by SP163M treatment (FIG. 4B). Thecardioprotective effects of SP16 translated in preservation of the leftventricular systolic function, measured as left ventricular fractionalshortening using transthoracic -echocardiography (FIG. 4C).

Protective effects of SP163M in Experimental Acute Myocardial Infarctionin the Mouse

Ten-weeks-old male ICR (CD1) mice underwent surgical coronary arteryligation for 30 minutes, followed by reperfusion for 24 hours, tosimulate the scenario of acute myocardial infarction (AMI) treated withreperfusion therapies. A subgroup of mice underwent sham surgery inwhich the coronary artery was identified and isolated but not ligated.

SP163M—active peptide—or a control solution—vehicle—were administered ata dose of 100 μg/mouse i.p. at reperfusion

Infarct size was measured at pathology with triphenyltetrazoliumchloride(TTC)/Evans blue staining, and by measuring plasma troponin Ilevels at 24 hours. Evans blue was retrogradely injected through theaorta after re-ligation of the coronary artery knot to identify thenon-risk myocardium (blue). TTC was used to stain viable myocardium(red).

Echocardiography was used to assess left ventricular (LV) fractionalshortening, as a measure of systolic function in vivo.

Each group included at least 6 mice.

Protective Effects of SP163M Administered Within 30 Minutes ofReperfusion in Experimental AMI in the Mouse

The option of delaying treatment with SP163M for additional 30 minutesafter reperfusion was examined. A control peptide SP34 which has 16/17amino acids identical in sequence as SP163M but in a scrambled order wasalso tested.

Ten-weeks-old male ICR (CD1) mice underwent surgical coronary arteryligation for 30 minutes, followed by reperfusion for 24 hours, tosimulate the scenario of AMI treated with reperfusion therapies.

SP163M—active peptide—or SP34—control peptide—were administered at adose of 100 μg/mouse i.p. at the moment of the reperfusion or with 30′of delay, to simulate a clinical scenario in which a delay betweenreperfusion and pharmacologic therapy is likely to occur.

Infarct size was measured with TTC/Evans blue staining. Echocardiographywas used to assess left ventricular (LV) fractional shortening, as ameasure of systolic function in vivo. Each group included at least 6mice.

SP163M given with 30′ of reperfusion significantly reduced infarct size(FIG. 5A) and preservation of LV fractional shortening (FIG. 5B).

SP163M provides a dose-dependent reduction in infarct size inexperimental AMI in the mouse

The cardio-protective capacity of SP163M at 10μg dose to the 100 μg dosewas compared. The results are depicted in FIG. 6 showing that even areduction of SP163M dose by 90% was shown to be effective as measured byinfarct size and LV systolic function (FIGS. 6A and 6B, respectively).

Ten-weeks-old ICR mice underwent surgical coronary artery ligation for30 minutes, followed by reperfusion for 24 hours. SP16 was administeredat 2 different doses (10 or 100 ug) with 30′ of delay. The controlpeptide SP34 (100 μg) or the vehicle solution were used as controls.

Infarct size was measured with TTC/Evans blue staining. Transthoracicechocardiography was used to assess left ventricular (LV) fractionalshortening. Each group included at least 6 mice.

SP163M given with 30′ of reperfusion reduces infarct size (FIG. 6A) andpreserves LV fractional shortening (FIG. 6B) in a dose-dependentfashion.

SP163M has No Effects on Cardiac Contractility in the Healthy Mouse

To test the effect of SP163M on heart contractility the following studywas conducted. SP163M peptide was shown to not effect heartcontractility which adds to the safety profile of this peptide drug(FIG. 7). SP34 is a peptide with a scrambled sequence of SP16 and isused as a control peptide.

Ten-weeks-old ICR mice were treated with SP163M (100 μg/mouse i.p.), thecontrol peptide SP34 (100 μg/mouse) or the vehicle solution.Echocardiography was used to assess left ventricular (LV) fractionalshortening at baseline and after 180 minutes.

Cardiac contractility reserve was tested with isoproterenol challenge(10 ng/kg i.p.), measuring the increase in LV fractional shorteningwithin 5′ of treatment. Each group included at least 6 mice.

SP163M does not alter cardiac systolic function at rest (FIG. 7, leftpanel) or after isoproterenol challenge (contractile reserve)(FIG. 7,right panel).

Discussion

In addition to being its anti inflammatory effect, SP163M is alsocardio-protective. It reduces the size of the infarct through rescuingthe cardiac cells from death upon reperfusion IL1 antagonist tested inAMI and showed a clear anti inflammatory effect but no reduction ininfarct size (not cardio-protective). SP163M cell-protective effectmanifested in AMI in a decrease of infarct can also be applicable toother injuries resulting in cell death such as other ischemiasituations, stroke, traumatic brain injury and toxic shocks. It iscontemplated herein that SP163M can permit treatment of Acute MyocardialInfarction, gout, stroke, complications associated with heart surgery,traumatic brain injury, any other disease with “cytokine storm”involvement.

REFERENCES

-   1. Velagaleti R S, Pencina M J, Murabito J M et al. Long-term trends    in the incidence of heart failure after myocardial infarction.    Circulation 2008; 118:2057-2062-   2. Eapen Z J, Tang W H, Felker G M et al. Defining heart failure end    points in ST-elevation myocardial infarction trials: integrating    past experiences to chart a path forward. Circ Cardiovasc Qual    Outcomes 2012; 5:594-600-   3. Mezzaroma E, Toldo S, Farkas D, et al. The inflammasome promotes    adverse cardiac remodeling following acute myocardial infarction in    the mouse. Proc Natl Acadi Scie USA 2011; 108:19725-19730-   4. Roubille F, Samri A, Comillet L, et al. Routinely-feasible    multiple biomarkers score to predict prognosis after revascularized    STEMI. Eur J Intern Med 2010; 21:131-136-   5. Lewis E C. Expanding the clinical indications for    α(1)-antitrypsin therapy. Mol Med 2012; 18:957-70.-   6. Toldo S, Seropian I M, Mezzaroma E, et al. Alpha-1 antitrypsin    inhibits caspase-1 and protects from acute myocardial    ischemia-reperfusion injury. J Mol Cell Cardiol 2011; 51:244-51.-   7. Pott G B, Chan E D, Dinarello C A, Shapiro L. Alpha-1-antitrypsin    is an endogenous inhibitor of proinflammatory cytokine production in    whole blood. J Leukoc Biol. 2009; 85:886-95.-   8. Abbate A, Salloum F N, Vecile E, et al. Anakinra, a recombinant    human interleukin-1 receptor antagonist, inhibits apoptosis in    experimental acute myocardial infarction. Circulation 2008;    117:2670-2683-   9. Abbate A, Kontos M C, Grizzard J D, et al. Interleukin-1 blockade    with anakinra to prevent adverse cardiac remodeling after acute    myocardial infarction (Virginia Commonwealth University Anakinra    Remodeling Trial [VCU-ART] Pilot Study). Am J Cardiol 2010;    15:1371-1377-   10. Abbate A, Van Tassell B W, Biondi-Zoccai G G, Kontos M C,    Grizzard J D, Spillman D W, Oddi C, Roberts C S, Melchior R D,    Mueller G H, Abouzaki N A, Rengel L R, Varma A, Gambill M L, Falcao    R A, Voelkel N F, Dinarello C A, Vetrovec G W. Effects of    Interleukin-1 Blockade with Anakinra on Adverse Cardiac Remodeling    and Heart Failure Following Acute Myocardial Infarction (from the    VCU-ART2 Pilot Study). Am J Cardiol 2013—in press.

Example 2

Met was replaced by Nle to enhance peptide stability, e.g., in thepeptide of SEQ ID NO:57-Ac-Val-Lys-Phe-Asn-Lys-Pro-Phe-Val-Phe-Leu-Nle-Ile-Glu-Gln-Asn-Thr-Lys-NH2(SEQ ID NO: 57). Methionine can be oxidized.

Example 3 LRP1 Mediates SP16-Induced Cardioprotection

SP163M is demonstrated herein to bind to LRP1 (Low density lipoproteinreceptor-related protein) and to be an agonist (as was shown for theparental AAT) of LRP1. It is further shown that blocking LRP1 eliminatesthe cardioprotective effects of SP163M and AAT.

Introduction

LRP1 is a receptor responsible for plasma clearance of SECs (SerpinEnzyme Complexes accumulating during an inflammatory response). Thus,LRP1's role is to block and discontinue inflammation. LRP1 can act viadirect modulation of key inflammatory pathways; e.g. NFkB, IRF-3, andAP-1 (e.g., ERKp42/p44, p38 and JNK).

In murine peritoneal macrophages, LRP1 down regulates the LPS driveninflammatory response through intracellular interaction with IRF-3.Additionally, LRP1 regulates inflammatory cytokines, impactsphagocytosis and cellular migration, and Treg profile. Serpins,including, e.g., the peptides of SEQ ID NO: 1 and 57, can bind toCluster II or IV of LRP1 via a highly conserved pentapeptide. See FIG.12A Accordingly, SP163M can induce a cascade leading to down-regulationof inflammatory response through cell signaling, modulation ofcytokines, decrease of CD4 T cells, and Th17 cells.

Modulation of LRP1 is implicated in a variety of inflammatory andauto-immune diseases. Diseases such as neurodegenerative diseases,atherosclerosis, and type II diabetes. Acute myocardial ischemia andreperfusion leads to myocardial necrosis and inflammation, which, ifleft unresolved can lead to tissue injury and heart failure. Ischemicinjury is the hallmark of AMI. Reperfusion, although beneficial inreducing the overall infarct size, is associated with more injury. Theinflammatory response to ischemia-reperfusion injury, while promotinginfarct healing, induces further loss of viable myocardium leading todysfunctional scar formation. A large infarct size, an intenseinflammatory response, and the formation of dysfunctional scar, allrepresent the substrate for heart failure following AMI. Promptresolution of the inflammatory response, on the other hand, isassociated with more favorable healing and reduced incidence of heartfailure.

Although not wishing to be bound by theory, it is proposed that theinflammatory response to tissue injury following AMI leads to therecruitment of plasma SERPINs at site of injury, inhibition of leukocyteserine proteases, and signaling toward resolution of the inflammatoryresponse through LRP1. It is therefore proposed that administration ofexogenous plasma-derived SERPINs early during AMI will lead toinhibition of the inflammatory injury through LRP1 resulting in acardioprotective effect and more prompt resolution of the inflammation.Furthermore, it is proposed that administration of a synthetic smallpeptide derived from the C3terminus of SERPINs will provide a powerfulLRP1 mediated cardioprotective signal, without inhibiting plasma serineproteas es. See FIG. 11.

SP163M Increases LRP1 Expression on Murine Macrophages

SP163M is demonstrated herein to increase expression of LRP1 on Raw264.7murine macrophages in a dose-dependent manner (FIG. 10). Macrophageswere plated 0.2×10{circumflex over ( )}6 in a 24 well plate and grown to70% confluence. Cells were treated with vehicle (red), SP16 at 50(Purple), 100 (Green) or 200 ug/ml (Blue), scrambled control (Orange) orCOG133 (ApoE peptide) (Pink) for 3 hrs before harvesting with cold PBS 5mM EDTA Samples were labeled with anti-LRP1 (abcam ab92544) and stainedwith Alexa Fluor™ 488 rabbit secondary (invitrogen) and then analyzed byflow cytometry (Guava easyCyte™ flow cytometer). The mean fluorescentintensity (MFI) normalized to the control is shown. (For eachsample>10,000 events were collected).

SP163M Binds to LRP1

An in vitro assay was performed to examine SP163M binding to LRP1. Toperform the assay, recombinant LRP1 protein (1 μg/ml) (R&D Systems,Minneapolis, Minn.) was incubated on ice in the presence ofbiotin-SP163M (1-5 μg/ml) at 4° C. for 1 hour. This mixture was thenincubated with 1 mg/ml M280 Streptavidin Dynabeads (Invitrogen,Carlsbad, Calif.) for 1.5 hours at 4° C. Dynabeads were washed inPBS+0.1% Tween20 and boiled in SDS loading buffer for 5 minutes. Thesupernatants were separated on NuPAGE 4-12% Bis-Tris gels (Invitrogen)and transferred to nitrocellulose membranes. The membranes were probedwith anti-LRP1 (LRP-1 Cluster II Affinity Purified Polyclonal Ab, R&D,Minneapolis, Minn.) and incubated overnight at 4° C. The membranes werethen washed 3 times with TBS-T and incubated with secondary HRP-coupledanti-goat diluted 1:10,000 in 5% milk TBS-T. The western blots werevisualized by chemiluminescence using SuperSignal West Femto MaximumSensitivity Substrate kit (ThermoScientific) and a Bio Rad MolecularImager ChemiDoc XRS system (Bio-Rad, Hercules, Calif.).

FIG. 12B shows that LRP1 binds SP163M at concentrations of 1 μg and 5μg. Meanwhile LRP1 did not bind a control, SP34, peptide.

SP163M is an LRP1 Agonist

THP1-XBlue™-MD2-CD14 cells (InvivoGen, San Diego, Calif.) were used inan in vitro assay to examine whether SP163M is an agonist of LRP1. TheTHP1-XBlue™-MD2-CD14 cells were maintained in RPMI 1640 medium (ThermoScientific) supplemented with 10% heat inactivated FBS, 1% Pen-Strep,100 ug/ml Normocin™, 200 ug/ml Zeocin™, and 250 μg/mlof G418. The celllines were maintained in 5% CO2 at 37° C. and in accordance with thedistributor's guidelines.

The THP1-XBlue™-MD2-CD14 cells were seeded at 1×10⁵ cells/well in a96-well cell culture plate. The cells were treated with peptide or ascrambled control peptide (100 μg/ml), for 30 minutes prior to theaddition of LPS (5 ng/ml) or GP96 (100 pM). After an 18-24 hourincubation at 37° C., 20 μl of supernatant was transferred to 180 μl ofQUANTI-Blue™ SEAP detection medium. NF-κB inducible SEAP levels weredetected by measuring the absorbance. In experiments blocking LRP1, theanti-LRP1 antibody (125 μg/ml) (clone 5A6, Molecular Innovations, Novi,Mich.) or Receptor-Associated Protein (RAP) (1 μM) (MolecularInnovations) were used 30 minutes prior to the addition of the peptide.

As shown in FIG. 13, SP163M inhibits NF-kB signaling induced by LPS(FIG. 13A) and GP96 (FIG. 13B). Treatment with an LRP antibody or RAP (aco-factor leading to LRP1 downregulation, limits SP163M-relatedinhibition in both LPS and GP96 treated cells.

LRP1 Mediates SP163M-Induced Cardioprotection

To examine the whether LRP1 mediates the cardioprotective effect ofSP163M, groups of mice were treated with an LRP1 blocking antibody priorto experimental AMI. The resulting infarct size and LV systolic functionwere measured

Adult out-bred male CD1 mice (10 weeks of age) supplied by HarlanSprague Dawley (Indianapolis, IN) were used in this study. A singleoperator (S. T.) skilled in coronary artery surgical ligation in miceperformed all surgeries. Experimental AMI was induced by transientcoronary artery ligation of the left anterior descending coronary arteryfor 30 minutes to induce ischemia of the anterior wall and the apex(visible as pallor) followed by reperfusion, and leading to an infarctinvolving approximately 15% of the left ventricle [1].

At the time of the surgeries, the mice were deeply sedated with sodiumpentobarbital (70-100 mg/kg), intubated and placed in the right lateraldecubitus. Aleft thoracotomy was performed followed by pericardiectomyand ligation of the proximal left coronary artery, followed byreperfusion. After closure of the thoracic access, the mice were left torecover for up to 1 week with unlimited access to food and water. Onlythe mice that showed evidence of ischemia at visual inspection duringsurgery and involving the whole apex were randomly assigned to differentpost treatment groups by an investigator (A.G.M) not involved in theadjudication of the endpoints of interest. The experiments wereconducted under the guidelines of laboratory animals for biomedicalresearch published by National Institutes of Health (revised 2011). Thestudy protocol was approved by the Institutional Animal Care and UseCommittee of the Virginia Commonwealth University.

Mice that received LRP1 blocking antibody as a pretreatment wereadministered the antibody 16 hours prior to the surgery to allowadequate time for antibody binding to the target receptor. Mice wererandomly assigned to various post-surgery treatment groups, where thepost-surgery treatment was given as a single intraperitonealadministration immediately after reperfusion. The post-surgery treatmentgroups were: SP163M 100 μg, matching volume of vehicle to SP163M, LRP1blocking antibody (AB) clone 5A6 3 mg/kg (Molecular Innovations),LRP1-AB and SP163M 100 μg, plasma derived alpha-1 antitrypsin (AAT) 60mg/kg (Aralast N P, Baxter, Deerfield, Ill., USA), LRP1-AB and AAT. Eachgroup included 5 to 8 mice.

Infarct size (reported in FIG. 14A) was measured as previously described[1]. A subgroup of mice were sacrificed 24 hours after surgery and thehearts were quickly removed and mounted on a Langendorff apparatus,where the coronary arteries were antegradely perfused with phosphatebuffered saline (PBS) 1× pH7.4, containing Heparin (40 U/ml). After theblood was washed out, 10% triphenyltetrazolium chloride (Sigma Aldrich)in PBS 1× was perfused, followed by the tying of the ligature andinfusion of 1% Phthalo blue dye (Quantum Ink, Louisville, Ky., USA) 5 mMadenosine in PBS1× was injected as a bolus into the aorta until most ofthe heart turned blue. After, the hearts were then removed from theLangendorff apparatus, frozen, and cut into 6 transverse slices of equalthickness, about 1 mm, from apex to base. The infarcted tissue(appearing white) and the viable tissue (bright red) were measured bycomputer morphometry using Image Pro Plus 6.0 software (MediaCybernetics, Silver Spring, MD). Infarct size is reported as % of theleft ventricle.

LV systolic function (reported in FIG. 14B) was determined as well. Themice underwent transthoracic echocardiography at baseline (beforesurgery) under mild anesthesia with sodium pentobarbital (30-50 mg/kg)and at 24 hours. Echocardiography was performed with the Vevo770 imagingsystem (VisualSonics Inc, Toronto, Ontario, Canada) and a 30-MHz probe[1, 2]. The heart was visualized in B-mode from parasternal short axisand apical views. We measured the left ventricular (LV) end-diastolicand end-systolic areas at B-Mode and the LV end-diastolic diameter(LVEDD), LV end-systolic diameters (LVESD) at M-Mode. LV fractionalshortening (FS) and LV ejection fraction (EF) were calculated. Asubgroup of mice underwent a repeated echocardiography at 7 days tomeasure the infarct size (number of segments with akinesis [2]) and theleft ventricular systolic function.

All results are expressed as mean and standard error. Comparisonsbetween 3 or more groups were performed using analysis of variance(ANOVA), followed by a T test for unpaired data to compare eachtreatment group with the respective control. Kaplan Meyer survivalcurves for SP16 and vehicle after LPS challenge were compared using theLog-rank test. We used Statistical Package for Social Sciences (SPSSversion 22.0, IBM inc., New York, N.Y.) for all analysis. A P value<0.05was considered significant.

Results. Treatment of the mice with blocking LRP1 antibody eliminatedthe protective effects of both SP163M and plasma-derived AAT.

REFERENCES

-   1. Toldo S, Seropian I M, Mezzaroma E, Van Tassell B W, Salloum F N,    Lewis E C, Voelkel N, Dinarello C A, Abbate A Alpha-1 antitrypsin    inhibits caspase-1 and protects from acute myocardial    ischemia-reperfusion injury. Journal of molecular and cellular    cardiology 2011, 51(2):244-251.-   2. Seropian I M, Abbate A, Toldo S, Harrington J, Smithson L,    Ockaili R, Mezzaroma E, Damilano F, Hirsch E, Van Tassell B W:    Pharmacologic Inhibition of Phosphoinositide 3-Kinase Gamma (PI3Kγ)    Promotes Infarct Resorption and Prevents Adverse Cardiac Remodeling    After Myocardial Infarction in Mice. Journal of cardiovascular    pharmacology 2010, 56(6):651-658.

1. A method of treating a disease associated with a cytokine storm,comprising administering to a human subject affected with the disease apharmaceutical composition comprising a peptide selected from the groupconsisting of: (a) a peptide comprising the amino acid sequence(SEQ ID NO: 1) VKFNKPFVFLMIEQNTK;

(b) a peptide consisting essentially of the amino acid sequence(SEQ ID NO: 4) RFNRPFLR.

(c) a peptide consisting essentially of the amino acid sequence of(SEQ ID NO: 8) RRRFNRPFLRRR.

(d) a peptide consisting essentially of the amino acid sequence of(SEQ ID NO: 1) VKFNKPFVFLMIEQNTK;

(e) a peptide consisting essentially of the amino acid sequence of(SEQ ID NO: 10) FNRPFL;

(f) a peptide comprising (SEQ ID NO: 57) VKFNKPFVFL[Nle]IEQNTK.

wherein the peptide has a size of 35 amino acids or less.
 2. The methodof claim 1, wherein the disease associated with a cytokine storm isselected from the group consisting of: acute myocardial infarction(AMI); gout; stroke; heart surgery complications; and traumatic braininjury.
 3. A method of reducing infarct size in a human subject in needof treatment therefor, comprising administering to the human subject apharmaceutical composition comprising a peptide selected from the groupconsisting of: (a) a peptide comprising the amino acid sequence(SEQ ID NO: 1) VKFNKPFVFLMIEQNTK;

(b) a peptide consisting essentially of the amino acid sequence(SEQ ID NO: 4) RFNRPFLR.

(c) a peptide consisting essentially of the amino acid sequence of(SEQ ID NO: 8) RRRFNRPFLRRR.

(d) a peptide consisting essentially of the amino acid sequence of(SEQ ID NO: 1) VKFNKPFVFLMIEQNTK;

(e) a peptide consisting essentially of the amino acid sequence ofFNRPFL (SEQ ID NO: 10); and (f) a peptide comprising (SEQ ID NO: 57)VKFNKPFVFL[Nle]IEQNTK.

wherein the peptide has a size of 35 amino acids or less.
 4. The methodof claim 3, wherein the human subject in need of a reduction in infarctsize is a subject in need of treatment for a disease selected from thegroup consisting of: acute myocardial infarction (AMI); ischemia;stroke; traumatic brain injury; and toxic shock.
 5. (canceled)
 6. Themethod of claim 1, wherein the pharmaceutical composition isadministered orally or subcutaneously to the human subject.
 7. Themethod of claim 1, wherein the pharmaceutical composition furthercomprises at least one second peptide or protein attached to the peptideto form a fusion protein.
 8. (canceled)
 9. The method of claim 7,wherein the at least one second peptide or protein is an epitope tag ora half-life extender or both.
 10. The method of claim 1, wherein thepeptide comprises one or more D-amino acids.
 11. (canceled) 12.(canceled)
 13. The method of claim 1, wherein the peptide consists of 22amino acid residues, 21 amino acid residues, or fewer.
 14. (canceled)15. (canceled)
 16. (canceled)
 17. The method of claim 3, wherein thepharmaceutical composition is administered orally or subcutaneously tothe human subject.
 18. The method of claim 3, wherein the pharmaceuticalcomposition further comprises at least one second peptide or proteinattached to the peptide to form a fusion protein.
 19. The method ofclaim 18, wherein the at least one second peptide or protein is anepitope tag or a half-life extender or both.
 20. The method of claim 3,wherein the peptide comprises one or more D-amino acids.
 21. The methodof claim 3, wherein the peptide consists of 22 amino acid residues, 21amino acid residues, or fewer.
 22. A pharmaceutical compositioncomprising a peptide comprising the amino acid sequence ofVKFNKPFVFL[Nle]IEQNTK (SEQ ID NO: 57), and a pharmaceutically acceptablecarrier, wherein the peptide has a size of 35 amino acids or less. 23.The pharmaceutical composition of claim 22, wherein the peptide consistsof 22 amino acid residues, 21 amino acid residues, or fewer.
 24. Thepharmaceutical composition of claim 22, wherein the composition isformulated into a dosage form suitable for oral, parenteral,intraperitoneal, rectal, subcutaneous, nasal, vaginal, inhalant, dermal,or ocular administration.
 25. The pharmaceutical composition of claim22, wherein the peptide is fused to an epitope tag, or a half-lifeextender.
 26. The pharmaceutical composition of claim 22, wherein thepeptide is modified by PEGylation.
 27. The pharmaceutical composition ofclaim 22, wherein the N-terminus of the peptide is acetylated, theC-terminus of the peptide is amidated, or both.